Nucleic acids encoding receptor recognition factor stat 3 and methods of use thereof

ABSTRACT

Receptor recognition factors exist that recognizes the specific cell receptor to which a specific ligand has been bound, and that may thereby signal and/or initiate the binding of the transcription factor to the DNA site. The receptor recognition factor is in one instance, a part of a transcription factor, and also may interact with other transcription factors to cause them to activate and travel to the nucleus for DNA binding. The receptor recognition factor appears to be second-messenger-independent in its activity, as overt perturbations in second messenger concentrations are of no effect. The concept of the invention is illustrated by the results of studies conducted with interferon (IFN)-stimulated gene transcription, and particularly, the activation caused by both IFNα and IFN-γ. Specific DNA and amino acid sequences for various human and murine receptor recognition factors are provided, as are polypeptide fragments of two of the ISGF-3 genes, and antibodies have also been prepared and tested. The polypeptides confirm direct involvement of tyrosine kinase in intracellular message transmission. Numerous diagnostic and therapeutic materials and utilities are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present Application is a Division of copending U.S. Ser. No. 08/820/754, filed Mar. 19, 1997, which is a Division of copending U.S. Ser. No. 08/212/185, filed Mar. 11, 1994 which is a Continuation-In-Part of U.S. Ser. No. 08/126,588 and U.S. Ser. No. 08/126,595 both filed Sep. 24, 1993, both now abandoned, which are both Continuations-In-Part of U.S. Ser. No. 07/980,498, filed Nov. 23, 1992, now abandoned, which is a Continuation-In-Part of U.S. Serial No. 07/854,296, filed Mar. 19, 1992, now abandoned, the disclosures of which are hereby incorporated by reference in their entireties. Applicants claim the benefits of these Applications under 35 U.S.C. § 120.

RELATED PUBLICATIONS

The Applicants are authors or co-authors of several articles directed to the subject matter of the present invention. (1) Darnell et al., "Interferon-Dependent Transcriptional Activation: Signal Transduction Without Second Messenger Involvement?" THE NEW BIOLOGIST, 2(10):1-4, (1990); (2) X. Fu et al., "ISGF3, The Transcriptional Activator Induced by Interferon α, Consists of Multiple Interacting Polypeptide Chains" PROC. NATL. ACAD. SCI. USA, 87:8555-8559 (1990); (3) D. S. Kessler et al., "IFNα Regulates Nuclear Translocation and DNA-Binding Affinity of ISGF3, A Multimeric Transcriptional Activator" GENES AND DEVELOPMENT, 4:1753 (1990). All of the above listed articles are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to intracellular receptor recognition proteins or factors(i.e. groups of proteins), and to methods and compositions including such factors or the antibodies reactive toward them, or analogs thereof in assays and for diagnosing, preventing and/or treating cellular debilitation, derangement or dysfunction. More particularly, the present invention relates to particular IFN-dependent receptor recognition molecules that have been identified and sequenced, and that demonstrate direct participation in intracellular events, extending from interaction with the liganded receptor at the cell surface to transcription in the nucleus, and to antibodies or to other entities specific thereto that may thereby selectively modulate such activity in mammalian cells.

BACKGROUND OF THE INVENTION

There are several possible pathways of signal transduction that might be followed after a polypeptide ligand binds to its cognate cell surface receptor. Within minutes of such ligand-receptor interaction, genes that were previously quiescent are rapidly transcribed (Murdoch et al., 1982; Larner et al., 1984; Friedman et al., 1984; Greenberg and Ziff, 1984; Greenberg et al., 1985). One of the most physiologically important, yet poorly understood, aspects of these immediate transcriptional responses is their specificity: the set of genes activated, for example, by platelet-derived growth factor (PDGF), does not completely overlap with the one activated by nerve growth factor (NGF) or tumor necrosis factor (TNF) (Cochran et al., 1983; Greenberg et al., 1985; Almendral et al., 1988; Lee et al., 1990). The interferons (IFN) activate sets of other genes entirely. Even IFNα and IFNγ, whose presence results in the slowing of cell growth and in an increased resistance to viruses (Tamm et al., 1987) do not activate exactly the same set of genes (Lamer et al., 1984; Friedman et al., 1984; Celis et al., 1987, 1985; Larner et al., 1986).

The current hypotheses related to signal transduction pathways in the cytoplasm do not adequately explain the high degree of specificity observed in polypeptide-dependent transcriptional responses. The most commonly discussed pathways of signal transduction that might ultimately lead to the nucleus depend on properties of cell surface receptors containing tyrosine kinase domains [for example, PDGF, epidermal growth factor (EGF), colony-stimulating factor (CSF), insulin-like growth factor-1 (IGF-1); see Gill, 1990; Hunter, 1990) or of receptors that interact with G-proteins (Gilman, 1987). These two groups of receptors mediate changes in the intracellular concentrations of second messengers that, in turn, activate one of a series of protein phosphokinases, resulting in a cascade of phosphorylations (or dephosphorylations) of cytoplasmic proteins.

It has been widely conjectured that the cascade of phosphorylations secondary to changes in intracellular second messenger levels is responsible for variations in the rates of transcription of particular genes (Bourne, 1988, 1990; Berridge, 1987; Gill, 1990; Hunter, 1990). However, there are at least two reasons to question the suggestion that global changes in second messengers participate in the chain of events leading to specific transcriptional responses dependent on specific receptor occupation by polypeptide ligands.

First, there is a limited number of second messengers (cAMP, diacyl glycerol, phosphoinositides, and Ca²⁺ are the most prominently discussed), whereas the number of known cell surface receptor-ligand pairs of only the tyrosine kinase and G-protein varieties, for example, already greatly outnumbers the list of second messengers, and could easily stretch into the hundreds (Gill, 1990; Hunter, 1990). In addition, since many different receptors can coexist on one cell type at any instant, a cell can be called upon to respond simultaneously to two or more different ligands with an individually specific transcriptional response each involving a different set of target genes. Second, a number of receptors for polypeptide ligands are now known that have neither tyrosine kinase domains nor any structure suggesting interaction with G-proteins. These include the receptors for interleukin-2 (IL-2) (Leonard et al., 1985), IFNα (Uze et al., 1990), IFNγ (Aguet et al., 1988), NGF (Johnson et al., 1986), and growth hormone (Leung et al., 1987). The binding of each of these receptors to its specific ligand has been demonstrated to stimulate transcription of a specific set of genes. For these reasons it seems unlikely that global intracellular fluctuations in a limited set of second messengers are integral to the pathway of specific, polypeptide ligand-dependent, immediate transcriptional responses.

In PCT International Publication No. WO 92/08740 published May 29, 1992 by the applicant herein, the above analysis was presented and it was discovered and proposed that a receptor recognition factor or factors, served in some capacity as a type of direct messenger between liganded receptors at the cell surface and the cell nucleus. One of the characteristics that was ascribed to the receptor recognition factor was its apparent lack of requirement for changes in second messenger concentrations. Continued investigation of the receptor recognition factor through study of the actions of the interferons IFNα and IFNγ has further elucidated the characteristics and structure of the interferon-related factor ISGF-3, and more broadly, the characterization and structure of the receptor recognition factor in a manner that extends beyond earlier discoveries previously described. It is accordingly to the presentation of this updated characterization of the receptor recognition factor and the materials and methods both diagnostic and therapeutic corresponding thereto that the present disclosure is directed.

SUMMARY OF THE INVENTION

In accordance with the present invention, receptor recognition factors have been further characterized that appear to interact directly with receptors that have been occupied by their ligand on cellular surfaces, and which in turn either become active transcription factors, or activate or directly associate with transcription factors that enter the cells' nucleus and specifically binds on predetermined sites and thereby activates the genes. It should be noted that the receptor recognition proteins thus possess multiple properties, among them: 1) recognizing and being activated during such recognition by receptors; 2) being translocated to the nucleus by an inhibitable process (eg. NaF inhibits translocation); and 3) combining with transcription activating proteins or acting themselves as transcription activation proteins, and that all of these properties are possessed by the proteins described herein.

A further property of the receptor recognition factors (also termed herein signal transducers and activators of transcription--STAT) is dimerization to form homodimers or heterodimers upon activation by phosphorylation of tyrosine. In a specific embodiment, infra, Stat91 and Stat84 form homodimers and a Stat91-Stat84 heterodimer. Accordingly, the present invention is directed to such dimers, which can form spontaneously by phophorylation of the STAT protein, or which can be prepared synthetically by chemically cross-linkng two like or unlike STAT proteins.

The receptor recognition factor is proteinaceous in composition and is believed to be present in the cytoplasm. The recognition factor is not demonstrably affected by concentrations of second messengers, however does exhibit direct interaction with tyrosine kinase domains, although it exhibits no apparent interaction with G-proteins. More particularly, as is shown in a co-pending, co-owned application entitled "INTERFERON-ASSOCIATED RECEPTOR RECOGNITION FACTORS, NUCLEIC ACIDS ENCODING THE SAME AND METHODS OF USE THEREOF," filed on even date herewith, the 91 kD human interferon (IFN) -γ factor, represented by SEQ ID NO:4 directly interacts with DNA after acquiring phosphate on tyrosine located at position 701 of the amino acid sequence.

The recognition factor is now known to comprise several proteinaceous substituents, in the instance of IFNα and IFNγ. Particularly, three proteins derived from the factor ISGF-3 have been successfully sequenced and their sequences are set forth in FIG. 1 (SEQ ID NOS:1, 2), FIG. 2 (SEQ ID NOS:3, 4) and FIG. 3 (SEQ. ID NOS.5, 6) herein. Additionally, a murine gene encoding the 91 kD protein (i.e., the murine homologue of the human protein having an amino acid sequence of SEQ ID NO:4) has been identified and sequenced. The nucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO:8) are shown in FIGS. 13A-13C.

In a further embodiment, murine genes encoding homologs of the recognition factor have been succefutay sequenced and cloned into plasmids. A gene in plasmid 13sf1 has the nucleotide sequence (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO:10) as shown in FIGS. 14A-14C. A gene in plasmid 19sf6 has the nucleotide sequence (SEQ ID NO:11) and deduced amino acid sequence (SEQ ID NO:12) shown in FIGS. 15A-15C.

It is particularly noteworthy that the protein sequence of FIG. 1 (SEQ ID NO:2) and the sequence of the proteins of FIGS. 2 (SEQ ID NO:4) and 3 (SEQ ID NO:6) derive, respectively, from two different but related genes. Moreover, the protein sequence of FIG. 13 (SEQ ID NO:8) derives from a murine gene that is analogous to the gene encoding the protein of FIG. 2 (SEQ ID NO:4). Of further note is that the protein sequences of FIGS. 14 (SEQ ID NO:10) and 15 (SEQ ID NO:12) derive from two genes that are different from, but related to, the protein of FIG. 13 (FIG ID NO:8). It is clear from these discoveries that a family of genes exists, and that further family members likewise exist. Accordingly, as demonstrated herein, by use of hybridization techniques, additional such family members will be found.

Further, the capacity of such family members to function in the manner of the receptor recognition factors disclosed, herein may be assessed by determining those ligand that cau se the phosphorylation of the particular family members.

In its broadest aspect, the present invention extends to a receptor recognition factor implicated in the transcriptional stimulation of genes in target cells in response to the binding of a specific polypeptide ligand to its cellular receptor on said target cell, said receptor recognition factor having the following characteristics:

a) apparent direct interaction with the ligand-bound receptor complex and activation of one or more transcription factors capable of binding with a specific gene;

b) an activity demonstrably unaffected by the presence or concentration of second messengers;

c) direct interaction with tyrosine kinase domains; and

d) a perceived absence of interaction with G-proteins.

In a further aspect, the receptor recognition (STAT) protein forms a dimer upon activation by phosphorylation.

In a specific example, the receptor recognition factor represented by SEQ ID NO:4 possesses the added capability of acting as a transcription factor and, in particular, as a DNA binding protein in response to interferon-γ stimulation. This discovery presages an expanded role for the proteins in question, and other proteins and like factors that have heretofore been characterized as receptor recognition factors. It is therefore apparent that a single factor may indeed provide the nexus between the liganded receptor at the cell surface and direct participation in DNA transcriptional activity in the nucleus. This pleiotypic factor has the following characteristics:

a) It interacts with an interferon-γ-bound receptor kinase complex;

b) It is a tyrosine kinase substrate; and

c) When phosphorylated, it serves as a DNA binding protein.

More particularly, the factor represented by SEQ ID NO:4 is interferon-dependent in its activity and is responsive to interferon stimulation, particularly that of interferon-γ. It has further been discovered that activation of the factor represented by SEQ ID NO:4 requires phosphorylation of tyrosine-701 of the protein, and further still that tyrosine phosphorylation requires the presence of a functionally active SH2 domain in the protein. Preferably, such SH2 domain contains an amino acid residue corresponding to an arginine at position 602 of the protein.

In a still further aspect, the present invention extends to a receptor recognition factor interactive with a liganded interferon receptor, which receptor recognition factor possesses the following characteristics:

a) it is present in cytoplasm;

b) it undergoes tyrosine phosphorylation upon treatment of cells with IFNα or IFNγ;

c) it activates transcription of an interferon stimulated gene;

d) it stimulates either an ISRE-dependent or a gamma activated site (GAS)-dependent transcription in vivo;

e) it interacts with IFN cellular receptors, and

f) it undergoes nuclear translocation upon stimulation of the IFN cellular receptors with IFN.

The factor of the invention represented by SEQ ID NO:4 appears to act in similar fashion to an earlier determined site-specific DNA binding protein that is interferon-γ dependent and that has been earlier called the γ activating factor (GAF). Specifically, interferon-γ-dependent activation of this factor occurs without new protein synthesis and appears within minutes of interferon-γ treatment, achieves maximum extent between 15 and 30 minutes thereafter, and then disappears after 2-3 hours. These further characteristics of identification and action assist in the evaluation of the present factor for applications having both diagnostic and therapeutic significance.

In a particular embodiment, the present invention relates to all members of the herein disclosed family of receptor recognition factors except the 91 kD protein factors, specifically the proteins whose sequences are represented by one or more of SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.

The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a receptor recognition factor, or a fragment thereof, that possesses a molecular weight of about 113 kD and an amino acid sequence set forth in FIG. 1 (SEQ ID NO:2); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 113 kD receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 1 (SEQ ID NO:1). In another embodiment, the receptor recognition factor has a molecular weight of about 91 kD and the amino acid sequence set forth in FIG. 2 (SEQ ID NO:4) or FIG. 13 (SEQ ID NO:8); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 91 kD receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 2 (SEQ ID NO:3) or FIG. 13 (SEQ ID NO:8). In yet a further embodiment, the receptor recognition factor has a molecular weight of about 84 kD and the amino acid sequence set forth in FIG. 3 (SEQ ID NO:6); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 84 kD receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 3 (SEQ ID NO:5). In yet another embodiment, the receptor recognition factor has an amino acid sequence set forth in FIG. 14 (SEQ ID NO:10); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding such receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 14 (SEQ ID NO:9). In still another embodiment, the receptor recognition factor has an amino acid sequence set forth in FIG. 15 (SEQ ID NO:12); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding such receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 15 (SEQ ID NO:11).

The human and murine DNA sequences of the receptor recognition factors of the present invention or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the receptor recognition factors. For example, the probes may be prepared with a variety of known vectors, such as the phage λ vector. The present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences set forth in FIGS. 1, 2, 3, 13, 14 and 15 (SEQ ID NOS:1, 3, 5, 7, 9, and 11, respectively). Correspondingly, the preparation of antisense RNA and ribozymes are included herein.

The present invention also includes receptor recognition factor proteins having the activities noted herein, and that display the amino acid sequences set forth and described above and selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12.

In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present receptor recognition factor(s), and more particularly, the complete DNA sequence determined from the sequences set forth above and in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11.

According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active animal or human receptor recognition factor.

The concept of the receptor recognition factor contemplates that specific factors exist for correspondingly specific ligands, such as tumor necrosis factor, nerve growth factor and the like, as described earlier. Accordingly, the exact structure of each receptor recognition factor will understandably vary so as to achieve this ligand and activity specificity. It is this specificity and the direct involvement of the receptor recognition factor in the chain of events leading to gene activation, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities.

The present invention naturally contemplates several means for preparation of the recognition factor, including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope. The isolation of the cDNA amino acid sequences disclosed herein facilitates the reproduction of the recognition factor by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.

The invention includes an assay system for screening of potential drugs effective to modulate transcriptional activity of target mammalian cells by interrupting or potentiating the recognition factor or factors. In one instance, the test drug could be administered to a cellular sample with the ligand that activates the receptor recognition factor, or an extract containing the activated recognition factor, to determine its effect upon the binding activity of the recognition factor to any chemical sample (including DNA), or to the test drug, by comparison with a control.

The assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the receptor recognition and/or transcription factors or proteins, either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating transcriptional activity. Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity. For example, such drugs might be used to modulate cellular response to shock, or to treat other pathologies, as for example, in making IFN more potent against cancer.

In yet a further embodiment, the invention contemplates antagonists of the activity of a receptor recognition factor (STAT). In particular, an agent or molecule that inhibits dimerization (homodimerization or heterodimerization) can be used to block transcription activation effected by an acitvated, phosphorylated STAT protein. In a specific embodiment, the antagonist can be a peptide having the sequence of a portion of an SH2 domain of a STAT protein, or the phophotyrosine domaine of a STAT protein, or both. If the peptide contains both regions, preferably the regions are located in tandem, more preferably with the SH2 domain portion N-terminal to the phosphotyrosine portion. In a specific example, infra, such peptides are shown to be capable of disrupting dimerization of STAT proteins.

One of the characteristics of the present receptor recognition factors is their participation in rapid phosphorylation and dephosphorylation during the course of and as part of their activity. Significantly, such phosphorylation takes place in an interferon-dependent manner and within a few minutes in the case of the ISGF-3 proteins identified herein, on the tyrosine residues defined thereon. This is strong evidence that the receptor recognition factors disclosed herein are the first true substrates whose intracellular function is well understood and whose intracellular activity depends on tyrosine kinase phosphorylation. In particular, the addition of phosphate to the tyrosine of a transcription factor is novel. This suggests further that tyrosine kinase takes direct action in the transmission of intracellular signals to the nucleus, and does not merely serve as a promoter or mediator of serine and/or serinine kinase activity, as has been theorieed to date. Also, the role of the factor represented by SEQ ID NO:2 in its activated phosphorylated form suggests possible independent therapeutic use for this activated form. Likewise, the role of the factor as a tyrosine kinase substrate suggests its interaction with kinase in other theatres apart from the complex observed herein.

The diagnostic utility of the present invention extends to the use of the present receptor recognition factors in assays to screen for tyrosine kinase inhibitors.

Because the activity of the receptor recognition-transcriptional activation proteins described herein must maintain tyrosine phosphorylation, they can and presumably are dephosphorylated by specific tyrosine phosphatases. Blocking of the specific phosphatase is therefore an avenue of pharmacological intervention that would potentiate the activity of the receptor recognition proteins.

The present invention likewise extends to the development of antibodies against the receptor recognition factor(s), including naturally raised and recombinantly prepared antibodies. For example, the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the receptor recognition factor(s). Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating transcriptional activity.

In particular, antibodies against specifically phosphorylated factors can be selected and are included within the scope of the present invention for their particular ability in following activated protein. Thus, activity of the recognition factors or of the specific polypeptides believed to be causally connected thereto may therefore be followed directly by the assay techniques discussed later on, through the use of an appropriately labeled quantity of the recognition factor or antibodies or analogs thereof.

Thus, the receptor recognition factors, their analogs and/or analogs, and any antagonists or antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the receptor recognition factor that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.

In an immunoassay, a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached. For example, antibodies against specifically phosphorylated factors may be selected and appropriately employed in the exemplary assay protocol, for the purpose of following activated protein as described above.

In the instance where a radioactive label, such as the isotopes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, and ¹⁸⁶ Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized calorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.

The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the recognition factors, or to identify drugs or other agents that may mimic or block their activity. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the recognition factors, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).

In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the recognition factor(s), its (or their) subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from the binding activity of the recognition factor or its subunits, and comprises administering an agent capable of modulating the production and/or activity of the recognition factor or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host. For example, drugs or other binding partners to the receptor recognition/transcription factors or proteins may be administered to inhibit or potentiate transcriptional activity, as in the potentiation of interferon in cancer therapy. Also, the blockade of the action of specific tyrosine phosphatases in the dephosphorylation of activated (phosphorylated) recognition/transcription factors or proteins presents a method for potentiating the activity of the receptor recognition factor or protein that would concomitantly potentiate therapies based on receptor recognition factor/protein activation.

More specifically, the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise effective inhibitors or enhancers of activation of the recognition factor or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention. For example, drugs or other binding partners to the receptor recognition/transcription factor or proteins, as represented by SEQ ID NO:2, may be administered to inhibit or potentiate transcriptional activity, as in the potentiation of interferon in cancer therapy. Also, the blockade of the action of specific tyrosine phosphatases in the dephosphorylation of activated (phosphorylated) recognition/transcription factor or protein presents a method for potentiating the activity of the receptor recognition factor or protein that would concomitantly potentiate therapies based on receptor recognition factor/protein activation. Correspondingly, the inhibition or blockade of the activation or binding of the recognition/transcription factor would affect MHC Class II expression and consequently, would promote immunosuppression. Materials exhibiting this activity, as illustrated later on herein by staurosporine, may be useful in instances such as the treatment of autoimmune diseases and graft rejection, where a degree of immunosuppression is desirable.

In particular, the proteins of ISGF-3 whose sequences are presented in SEQ ID NOS:2, 4, 6, 8, 10 or 12 herein, their antibodies, agonists, antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances wherein interferon therapy is appropriate, such as to treat chronic viral hepatitis, hairy cell leukemia, and for use of interferon in adjuvant therapy. The specificity of the receptor proteins hereof would make it possible to better manage the aftereffects of current interferon therapy, and would thereby make it possible to apply interferon as a general antiviral agent.

Accordingly, it is a principal object of the present invention to provide a receptor recognition factor and its subunits in purified form that exhibits certain characteristics and activities associated with transcriptional promotion of cellular activity.

It is a further object of the present invention to provide antibodies to the receptor recognition factor and its subunits, and methods for their preparation, including recombinant means.

It is a further object of the present invention to provide a method for detecting the presence of the receptor recognition factor and its subunits in mammals in which invasive, spontaneous, or idiopathic pathological states are suspected to be present.

It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity or combating the adverse effects of the recognition factor and/or its subunits in mammals.

It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the recognition factor or subunits thereof, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.

It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the recognition factor or its subunits, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states.

It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the recognition factor, its subunits, their binding partner(s), or upon agents or drugs that control the production, or that mimic or antagonize the activities of the recognition factors.

Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E depicts the full receptor recognition factor nucleic acid sequence and the deduced amino acid sequence derived for the ISGF-3α gene defining the 113 kD protein. The nucleotides are numbered from 1 to 2553 (SEQ ID NO:1), and the amino acids are numbered from 1 to 851 (SEQ ID NO:2).

FIGS. 2A-2E depicts the full receptor recognition factor nucleic acid sequence and the deduced amino acid sequence derived for the ISGF-3α gene defining the 91 kD protein. The nucleotides are numbered from 1 to 3943 (SEQ ID NO:3), and the amino acids are numbered from 1 to 750 (SEQ ID NO:4).

FIGS. 3A-3D depicts the full receptor recognition factor nucleic acid sequence and the deduced amino acid sequence derived for the ISGF-3α gene defining the 84 kD protein. The nucleotides are numbered from 1 to 2166 (SEQ ID NO:5), and the amino acids are numbered from 1 to 712 (SEQ ID NO:6).

FIG. 4 shows the purification of ISGF-3. The left-hand portion of the Figure shows the purification of ISGF-3 demonstrating the polypeptides present after the first oligonucleotide affinity column (lane 3) and two different preparations after the final chromatography step (Lanes 1 and 2). The left most lane contains protein size markers (High molecular weight, Sigma). ISGF-3 component proteins are indicated as 113 kD, 91 kD, 84 kD, and 48 kD [Kessler et al., GENES & DEV, 4 (1990); Levy et al., THE EMBO. J., 9 (1990)]. The right-hand portion of the Figure shows purified ISGF-3 from 2-3×10¹¹ cells was electroblotted to nitrocellulose after preparations 1 and 2 (Lanes 1 and 2) had been pooled and separated on a 7.5% SDS polyacrylamide gel. ISGF-3 component proteins are indicated. The two lanes on the right represent protein markers (High molecular weight, and prestained markers, Sigma).

FIGS. 5A-5B generally presents the results of Northern Blot analysis for the 91/84 kD peptides. FIG. 5a presents restriction maps for cDNA clones E4 (top map) and E3 (bottom map) showing DNA fragments that were radiolabeled as probes (probes A-D). FIG. 5b comprises Northern blots of cytoplasmic HeLa RNA hybridized with the indicated probes. The 4.4 and 3.1 KB species as well as the 28S and 18S rRNA bands are indicated.

FIG. 6 depicts the conjoint protein sequence of the 91 kD (SEQ ID NO:4) and 84 kD (SEQ ID NO:6) proteins of ISGF-3. One letter amino acid code is shown for the open reading frame from clone E4, (encoding the 91 kD protein). The 84 kD protein, encoded by a different cDNA (E3), has the identical sequence but terminates after amino acid 712, as indicated. Tryptic peptides t19, t13a, and t13b from the 91 kD protein are indicated. The sole recovered tryptic peptide from the 84 kD protein, peptide t27, was wholly contained within peptide t19 as indicated.

FIGS. 7A and 7B presents the results of Western blot and antibody shift analyses.

a) Highly purified ISGF-3, fractionated on a 7.0% SDS polyacrylaride gel, was probed with antibodies a42 (amino acids 597-703); a55 (amino acids 2-59); and a57 (amino acids 705-739) in a Western blot analysis. The silver stained part of the gel (lanes a, b, and c) illustrates the location of the ISGF-3 component proteins and the purity of the material used in Western blot: Lane a) Silver stain of protein sample used in all the Western blot experiments (immune and preimmune). Lane b) Material of equal purity to that shown in FIG. 4, for clearer identification of the ISGF-3 proteins. Lane c) Size protein markers indicated.

b) Antibody interference of the ISGF-3 shift complex; Lane a) The complete ISGF-3 and the free ISGF-3γ component shift with partially purified ISGF-3 are marked; Lane b) Competition with a 100 fold excess of cold ISRE oligonucleotide. Lane c) Shift complex after the addition of 1 ml of preimmune serum to a 12.5 μl shift reaction. Lanes d and e)--Shift complex after the addition of 1 μl of a 1:10 dilution or 1 ml of undiluted a42 antiserum to a 12.5 μl shift reaction.

Methods:

Antibodies a42, a55 and a57 were prepared by injecting approximately 500 mgm of a fusion protein prepared in E. coli using the GE3-3X vector [Smith et al., GENE, 67 (1988)]. Rabbits were bled after the second boost and serum prepared.

For Western blots highly purified ISGF-3 was separated on a 7% SDS polyacrylamide gel and electroblotted to nitrocellulose. The filter was incubated in blocking buffer ("blotto"), cut into strips and probed with specific antiserum and preimmune antiserum diluted 1:500. The immune complexes were visualized with the aid of an ECL kit (Amersham). Shift analyses were performed as previously described [Levy et al., GENES & DEV., 2 (1988); Levy et al., GENES & DEV., 3 (1989)] in a 4.5% polyacrylamide gel.

FIG. 8 presents the full length amino acid sequence of 113 kD protein components of ISGF-3α (SEQ ID NO:2) and alignment of conserved amino acid sequences between the 113 kD and 91/84 kD proteins (SEQ ID NOS:4 AND 6).

A. Polypeptide sequences (A-E) derived from protein micro-sequencing of purified 113 kD protein (see accompanying paper) are underlined. Based on peptide E, we designed a degenerate oligonucleotide, AAT/CACIGAA/GCCIATGGAA/GATT/CATT (SEQ ID NO:13), which was used to screen a cDNA library [Pine et al., MOL. CELL. BIOL., 10 (1990)] basically as described [Norman et al., CELL, 55 (1988)]. Briefly, the degenerate oligonucleotides were labeled by 32P-γ-ATP by polynucleotide kinase, hybridizations were carried out overnight at 40° C. in 6×SSTE (0.9 M NaCl, 60 mM Tris-HCl [pH 7.9 ] 6 mM EDTA), 0.1% SDS, 2 mM Na₂ P₅ O₇, 6 mM KH₂ PO₄ in the presence of 100 mg/ml salmon sperm DNA sperm and 10×Denhardt's solution [Maniatis et al., MOLECULAR CLONING; A LABORATORY MANUAL (Cold Spring Harbor Lab., 1982)]. The nitrocellulose filters then were washed 4×10 min. with the same hybridization conditions without labeled probe and salmon sperm DNA. Autoradiography was carried out at -80° C. with intensifying screen for 48 hrs. A PCR product was obtained later by the same method described for the 91/84 kD sequences, by using oligonucleotides designed according polypeptide D and E. The sequence of this PCR product was identical to a region in clone f11. The full length of 113 kD protein contains 851 amino acids. Three major helices in the N-terminal region were predicted by the methods of both Chou and Fasman [Chou et al., ANN. REV. BIOCHEM., 47 (1978)] and Garnier et al [Garnier et al., J. MOL. BIOL., 12 (1978)] and are shown in shadowed boxes. At the C-terminal end, a highly negative charged domain was found. All negative charged residues are blackened and positive charged residues shadowed. The five polypeptides that derived from protein microscreening [Aebersold et al., PROC. NATL. ACAD. SCI. USA, 87 (1987)] are underlined.

B) Comparison of amino acid sequences of 113 kD and 91/84 kD protein shows a 42% identical amino acid residues in the overlapping 715 amino acid sequence shown. In the middle helix region four leucine and one valine heptad repeats were identified in both 113 and 91/84 kD protein (the last leucine in 91/84 kD is not exactly preserved as heptad repeats). When a heligram structure was drawn this helix is amphipathic (not shown). Another notable feature of this comparison is several tyrosine residues that are conserved in both proteins near their ends.

FIG. 9 shows the in vitro transcription and translation of 113 kD and 91 kD cDNA and a Northern blot analysis with 113 kD cDNA probe.

a) The full length cDNA clones of 113 and 91 kD protein were transcribed in vitro and transcribed RNAs was translated in vitro with rabbit lenticulate lysate (Promega; conditions as described in the Promega protocol). The mRNA of BMV (Promega) was simultaneously translated as a protein size marker. The 113 cDNA yielded a translated product about 105 kD and the 91 cDNA yielded a 86 kD product.

b) When total cytoplasmic mRNAs isolated from superinduced HeLa cells were utilized, a single 4.8 KB mRNA band was observed with a cDNA probe coding for C-end of 113 kD protein in a Northern blot analysis [Nielsch et al., The EMBO. J., 10 (1991)].

FIG. 10(A) presents the results of Western blot analysis confirming the identity of the 113 kD protein. An antiserum raised against a polypeptide segment [Harlow et al., ANTIBODIES; A LABORATORY MANUAL (Cold Spring Harbor Lab., 1988)] from amino acid 500 to 650 of 113 kD protein recognized specifically a 113 kD protein in a protein Western blot analysis. The antiserum recognized a band both in a highly purified ISGF-3 fraction (>10,000 fold) from DNA affinity chromatography and in the crude extracts prepared from γ and α IFN treated HeLa cells [Fu et al., PROC. NATL. ACAD. SCI. USA, 87 (1990)]. The antiserum was raised against a fusion protein [a cDNA fragment coding for part of 113 kD protein was inserted into pGEX-2T, a high expression vector in the E. coli [Smith et al., PROC. NATL. ACAD. SCI. USA, 83 (1986)] purified from E. coli [Smith et al., GENE, 67 (1988)]. The female NZW rabbits were immunized with 1 mg fusion protein in Freund's adjuvant. Two subsequent boosts two weeks apart were carried out with 500 mg fusion protein. The Western blot was carried out with conditions described previously [Pine et al., MOL. CELL. BIOL., 10 (1990)].

FIG. 10(B) presents the results of a mobility shift assay showing that the anti-113 antiserum affects the ISGF-3 shift complex. Preimmune serum or the 113 kD antiserum was added to shift reaction carried out as described [Fu et al. PROC. NATL. ACAD. SCI. USA, 87 (1990); Kessler et al. GENES & DEV., 4, (1990)] at room temperature for 20 min. then one-third of reaction material was loaded onto a 5% polyacrylamide gel. In addition unlabeled probe was included in one reaction to show specificity of the gel shift complexes.

FIG. 11 shows the results of experiments investigating the IFN-α dependent phosphorylation of 113, 91 and 84 kD proteins. Protein samples from cells treated in various ways after 60 min. exposure to ³² PO₄ ⁻³ were precipitated with antiserum to 113 kD protein. Lane 1, no treatment of cells; Lane 2, cells treated 7 min. with IFN-α. By comparison with the marker proteins labeled 200, 97.5, 69 and 46 kD (kilo daltons), the PO₄ ³¹ 3 labeled proteins in the precipitate are seen to be 113 and 91 kD. Lane 3, cells treated with IFN-γ overnight (no phosphorylated proteins) and then (Lane 4) treated with IFN-α for 7 min. show heavier phosphorylation of 113, 91 and 84 kD.

FIG. 12 is a chromatogram depicting the identification of phosphoamino acid. Phosphate labeled protein of 113, 91 or 84 kD size was hydrolyzed and chromatographed to reveal newly labeled phosphotyrosine. Cells untreated with IFN showed only phosphoserine label. (P Ser=phosphoserine; P Thr=phosphothreonine; P Tyr=phosphotyrosine.

FIG. 13 depicts (A) the deduced amino acid sequence (SEQ ID NO:8) of and (B-C) the DNA sequence (SEQ ID NO:7) encoding the murine 91 kD intracellular receptor recognition factor.

FIG. 14 depicts (A) the deduced amino acid sequence (SEQ ID NO:10) of and (B-D) the DNA sequence (SEQ ID NO:9) encoding the 13sf1 intracellular receptor recognition factor.

FIG. 15 depicts (A) the deduced amino acid sequence (SEQ ID NO:12) of and (B-E) the DNA sequence (SEQ ID NO:11) encoding the 19sf6 intracellular receptor recognition factor.

FIG. 16. Determination of molecular weights of Stat91 and phospho Stat91 by native gel analysis.

A) Western blot analysis of fractions from affinity purification. Extracts from human FS2 fibroblasts treated with IFN-γ (Ext), the unbound fraction (Flow), the fraction washed with Buffer AO.2 (AO.2), and the bound fraction eluted with buffer AO.8(AO.8) were immunoblotted with anti-91T.

B) Native gel analysis. Phosphorylated Stat91 (the AO.8 fraction from A) and unphosphorylated Stat91 (the Flow fraction from A) were analyzed on 4.5%, 5.5%, 6.5% and 7.5% native polyacrylamide gels followed by immunoblotting with anti-91T. The top of gels (TOP) and the migration position of bromophenol blue (BPB) are indicated.

C) Ferguson plots. The relative mobilities (Rm) of the Stat91 and phospho Stat91 were obtained from FIG. 1B (see Experimental Procedures). Closed circle:

Chicken egg albumin (45 kD); Cross: Bovine serum albumin, monomer (66 kD); Open square: Bovine serum albumin, dimer (132 kD); Open circle: Urease, trimer (272 kD); Open triangle: Unphosphorylated Stat91; Closed triangle: Phosphorylated Stat91.

D) Determination of molecular weights from the standard curve. The molecular weights of phosphorylated and unphosphorylated Stat91 proteins (indicated as closed and open arrows, respectively) were obtained by extrapolation of their retardation coefficients.

FIG. 17. Determination of molecular weights by glycerol gradients.

A) Western blot analysis. Extracts from human Bud8 fibroblasts treated with IFN-γ (the rightmost lane) and every other fraction from fraction 16 to 34 were analyzed on 7.5% SDS-PAGE followed by immunobloting with anti-91T. The peak of phosphorylated Stat91 (fraction 20) and the peak of unphosphorylated Stat91 (fraction 30) were indicated by a closed and open arrow, respectively.

B) Mobility shift analysis. Every other fractions from the gradients were analyzed.

C) Graphic representation of the data from A and B. Peak fraction numbers of protein standards are plotted versus their molecular weight. The position of peaks (of phosphorylated and unphosphorylated Stat91 protein are indicated by the closed and open arrows, respectively. Standards are ferritin (Fer, 440 kD), catalase (Cat, 232 kD), ferritin half unit (Fer 1/2, 220 kD), aldolase (Ald, 158 kD), bovine serum albumin (BSA, 68 kD).

FIG. 18. Stat91 in cell extracts binds DNA as a dimer.

A) Wester blot analysis. Extracts from stable cell lines expressing either Stat84 (C84), or Stat91L (C91L) or both (Cmx) were analyzed on 7.5% SDS-PAGE followed by immunobloting with anti-91.

B) Gel mobility shift analysis. Extracts from stable cell lines (FIG. 3A) untreated (-) or treated with IFN-γ(+) were analyzed. The positions of Stat91 homodimer (91L), Stat84 homodimer (84), and the heterodimer (84*91) are indicated.

FIG. 19. Formation of herterodimer by denaturation and renaturation. Cytoplasmic (Left Panel) or nuclear extracts (Right Panel) from IFN-γ-treated cell lines expressing either Stat84 (C84) or Stat91 (C91) were analyzed by gel mobility shift assays. +: with addition; -: without addition; D/R: samples were subjected to guanidinium hydrochloride denaturation and renaturation treatment.

FIG. 20. Diagramatic representation of dissociation and reassociation analysis.

FIG. 21. Dissociation-reassociation analysis with peptides. Gel mobility shift analysis with IFN-γ treated nuclear extracts from cell lines expressing Stat91L (C91L, lane 15) or Stat84 (C84, lane 14) or mixture of both (lane 1-13, 16-18) in the presence of increasing concentrations of various peptides. 91-Y, unphosphorylated peptide from Stat91 (LDGPKGTGYIKTELI) (SEQ. ID NO.:18); 91Y-p, phosphotyrosyl peptide from Stat91 (GY*IKTE) (SEQ ID NO.:19); 113Y-p, phosphotyrosyl peptide with high binding affinity to Src SH2 domain (EPQY*EEIPIYL, Songyang et al., 1993, Cell 72:767-778) (SEQ. ID NO.:21). Final concentrations of peptides added: 1 μM (lane 8), 4 μM (lane 2,5, 11), 10 μM (lane 9), 40 μM (lane 3, 6, 10, 12, 14-18), 160 μM (lane 4, 7, 13). +: with addition; -: without addition. Right panel: antiserum tests for identity of gel-shift bands (see FIG. 3).

FIG. 22. Dissociation-reassociation analysis with GST fusion proteins. A) SDS-PAGE (12%) analysis of purified GST fusion proteins as visualized by Commasie blue. GST-91 SH3, native SH2 domain of Stat91; GST-91 mSH2, R⁶⁰² to L⁶⁰² mutant; GST-91 SH3, SH3 domain of Stat91; GST Src SH2, the SH2 domain of src protein. Same amounts (1 μg) of each fusion proteins were loaded. Protein markers were run in lane 1 as indicated.

B) Dissociation-reassociation analysis similar to FIG. 6. Dissociating agents were GST fusion proteins purified from bacterial expression as shown above. Final concentrations of fusion proteins added are 0.5 μM (lanes 2, 5, 8, 11, 14), 2.5 μM (lanes 3, 6, 9, 12, 15) and 5 μM (lanes 4, 7, 10, 13, 17, 18). +: with addition; -: without addition; FP: fusion proteins.

FIG. 23. Comparison of Stat91 SH2 structure with known SH2 structures. The Stat91 sequence is disclosed herein (SEQ ID NO:4). The structures used for the other SH2s are Src (Waksman et al., 1992, Nature 358:646-653) (SEQ ID NO:22), AbI (Overduin et al., 1992, Proc. Natl. Acad. Sci. USA 89:11673-77 and 1992, Cell 70:697-704) (SEQ ID NO:23, Lck (Eck et al., 1993, Nature 362:87-91) (SEQ ID NO:24), and p85αN (Booker et al., 1992, Nature 358:684-687) (SEQ ID NO:25). The alignment of the determined structures is by direct coordinate superimposition of the backbone structures. The names of secondary structural features and significant residues is based on the scheme of Eck et al., 1993. The boundaries and extents of the structure features are indicated by [- - -]. The starting numbers for the parent sequences are shown in parentheses. Experimentally determined structurally conserved regions are from Src, p85α, and AbI (Cowburn, unpublished). The root mean square deviation of three-dimensionally aligned structures differs by less than 1 Angstrom for the backbone non-hydrogen atoms in the sections marked by the XXX.

DETAILED DESCRIPTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins eds. (1985)]; "Transcription And Translation" [B. D. Hames & S. J. Higgins, eds. (1984)]; "Animal Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Therefore, if appearing herein, the following terms shall have the definitions set out below.

The terms "receptor recognition factor", "receptor recognition-tyrosine kinase factor", "receptor recognition factor/tyrosine kinase substrate", "receptor recognition/transcription factor", "recognition factor"and "recognition factor protein(s)" and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in FIG. 1 (SEQ ID NO:2), FIG. 2 (SEQ ID NO:4) and in FIG. 3 (SEQ ID NO:6), and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms "receptor recognition factor", "recognition factor" and "recognition factor protein(s)" are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

    ______________________________________                                         TABLE OF CORRESPONDENCE                                                        SYMBOL                                                                         1-Letter  3-Letter        AMINO ACID                                           ______________________________________                                         Y         Tyr             tyrosine                                             G         Gly             glycine                                              F         Phe             phenylalanine                                        M         Met             methionine                                           A         Ala             alanine                                              S         Ser             serine                                               I         Ile             isoleucine                                           L         Leu             leucine                                              T         Thr             threonine                                            V         Val             valine                                               P         Pro             proline                                              K         Lys             lysine                                               H         His             histidine                                            Q         Gln             glutamine                                            E         Glu             glutamic acid                                        W         Trp             tryptophan                                           R         Arg             arginine                                             D         Asp             aspartic acid                                        N         Asn             asparagine                                           C         Cys             cysteine                                             ______________________________________                                    

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

The term "oligonucleotide", as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.

The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab')₂ and F(v), which portions are preferred for use in the therapeutic methods described herein.

Fab and F(ab')₂ portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab' antibody molecule portions are also well-known and are produced from F(ab')₂ portions followed by reduction of the disulfide bonds linkng the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.

The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the S phase activity of a target cellular mass, or other feature of pathology such as for example, elevated blood pressure, fever or white cell count as may attend its presence and activity.

A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5×SSC and 65° C. for both hybridization and wash.

In its primary aspect, the present invention concerns the identification of a receptor recognition factor, and the isolation and sequencing of a particular receptor recognition factor protein, that is believed to be present in cytoplasm and that serves as a signal transducer between a particular cellular receptor having bound thereto an equally specific polypeptide ligand, and the comparably specific transcription factor that enters the nucleus of the cell and interacts with a specific DNA binding site for the activation of the gene to promote the predetermined response to the particular polypeptide stimulus. The present disclosure confirms that specific and individual receptor recognition factors exist that correspond to known stimuli such as tumor necrosis factor, nerve growth factor, platelet-derived growth factor and the like. Specific evidence of this is set forth herein with respect to the interferons α and γ IFNα and IFNγ).

A further property of the receptor recognition factors (also termed herein signal transducers and activators of transcription--STAT) is dimerization to form homodimers or heterodimers upon activation by phosphorylation of tyrosine. In a specific embodiment, infra, Stat91 and Stat84 form homodimers and a Stat91-Stat84 heterodimer. Accordingly, the present invention is directed to such dimers, which can form spontaneously by phophorylation of the STAT protein, or which can be prepared synthetically by chemically cross-lining two like or unlike STAT proteins.

The present receptor recognition factor is likewise noteworthy in that it appears not to be demonstrably affected by fluctuations in second messenger activity and concentration. The receptor recognition factor proteins appear to act as a substrate for tyrosine kinase domains, however do not appear to interact with G-proteins, and therefore do not appear to be second messengers.

A particular receptor recognition factor identified herein by SEQ ID NO:4, has been determined to be present in cytoplasm and serves as a signal transducer and a specifice transcription factor in response to IFN-γ stimulation that enters the nucleus of the cell and interacts directly with a specific DNA binding site for the activation of the gene to promote the predetermined response to the particular polypeptide stimulus. This particular factor also acts as a translation protein and, in particular, as a DNA binding protein in response to interferon-γ stimulation. This factor is likewise noteworthy in that it has the following characteristics:

a) It interacts with an interferon-γ-bound receptor kinase complex;

b) It is a tyrosine kinase substrate; and

c) When phosphorylated, it serves as a DNA binding protein.

More particularly, the factor of SEQ ID NO:4 directly interacts with DNA after acquiring phosphate on tyrosine located at position 701 of the amino acid sequence. Also, interferon-γ-dependent activation of this factor occurs without new protein synthesis and appears within minutes of interferon-γ treatment, achieves maximum extent between 15 and 30 minutes thereafter, and then disappears after 2-3 hours.

In a particular embodiment, the present invention relates to all members of the herein disclosed family of receptor recognition factors except the 91 kD protein factors, specifically the proteins whose sequences are represented by one or more of SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.

Subsequent to the filing of the initial applications directed to the present invention, the inventors have termed each member of the family of receptor recognition factors as a signal transducer and activator of transcription (STAT) protein. Each STAT protein is designated by the apparent molecular weight (e.g., Stat 113, Stat91, Stat84, etc.), or by the order in which it has been identified (e.g., Stat1α [Stat91], Stat1β [Stat84], Stat2 [Stat113], Stat3 [a murine protein described in U.S. application Ser. No. 08/126,588, filed Sep. 24, 1993 as 19sf6], and Stat4 [a murine STAT protein described in U.S. application Ser. No. 08/126,588, filed Sep. 24, 1993 as 13sf1]). As will be readily appreciated by one of ordinary skill in the art, the choice of name has no effect on the intrinsic characteristics of the factors described herein, which were first disclosed in U.S. application Ser. No. 07/845,296, filed Mar. 19, 1992. The present inventors have chosen to adopt this newly derived terminology herein as a convenience to the skilled artisan who is familiar with the subsequently published papers relating to the same, and in accordance with the proposal to harmonize the terminology for the novel class of proteins, and nucleic acids encoding the proteins, disclosed by the instant inventors. The terms [molecular weight] kd receptor recognition factor, Stat[molecular weight], and Stat[number] are used herein interchangeably, and have the meanings given above. For example, the terms 91 kd protein, Stat91, and Stat1α refer to the same protein, and in the appropriate context refer to the nucleic acid molecule encoding such protein.

As stated above, the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a receptor recognition factor, or a fragment thereof, that possesses a molecular weight of about 113 kD and an amino acid sequence set forth in FIG. 1 (SEQ ID NO:2); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 113 kD receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 1 (SEQ ID NO:1). In another embodiment, the receptor recognition factor has a molecular weight of about 91 kD and the amino acid sequence set forth in FIG. 2 (SEQ ID NO:4) or FIG. 13 (SEQ ID NO:8); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 91 kD receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 2 (SEQ ID NO:3) or FIG. 13 (SEQ ID NO:8). In yet a further embodiment, the receptor recognition factor has a molecular weight of about 84 kD and the amino acid sequence set forth in FIG. 3 (SEQ ID NO:6); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 84 kD receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 3 (SEQ ID NO:5). In yet another embodiment, the receptor recognition factor has an amino acid sequence set forth in FIG. 14 (SEQ ID NO:10); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding such receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 14 (SEQ ID NO:9). In still another embodiment, the receptor recognition factor has an amino acid sequence set forth in FIG. 15 (SEQ ID NO:12); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding such receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 15 (SEQ ID NO:11).

The possibilities both diagnostic and therapeutic that are raised by the existence of the receptor recognition factor or factors, derive from the fact that the factors appear to participate in direct and causal protein--protein interaction between the receptor that is occupied by its ligand, and those factors that thereafter directly interface with the gene and effect transcription and accordingly gene activation. As suggested earlier and elaborated further on herein, the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the receptor recognition factor is implicated, to modulate the activity initiated by the stimulus bound to the cellular receptor.

Thus, in instances where it is desired to reduce or inhibit the gene activity resulting from a particular stimulus or factor, an appropriate inhibitor of the receptor recognition factor could be introduced to block the interaction of the receptor recognition factor with those factors causally connected with gene activation. Correspondingly, instances where insufficient gene activation is taking place could be remedied by the introduction of additional quantities of the receptor recognition factor or its chemical or pharmaceutical cognates, analogs, fragments and the like.

As discussed earlier, the recognition factors or their binding partners or other ligands or agents exhibiting either mimicry or antagonism to the recognition factors or control over their production, may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated specific transcriptional stimulation for the treatment thereof. A variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the recognition factors or their subunits may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the recognition factors and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as viral infection or the like. For example, the recognition factor or its subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Likewise, small molecules that mimic or antagonize the activity(ies) of the receptor recognition factors of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.

The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques" (1980); Hammerling et al., "Monoclonal Antibodies And T-cell Hybridomas" (1981); Kennett et al., "Monoclonal Antibodies" (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against recognition factor peptides can be screened for various properties; i.e., isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that neutralize the activity of the recognition factor or its subunits. Such monoclonals can be readily identified in recognition factor activity assays. High affinity antibodies are also useful when immunoaffinity purification of native or recombinant recognition factor is possible.

Preferably, the anti-recognition factor antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb). In addition, it is preferable for the anti- recognition factor antibody molecules used herein be in the form of Fab, Fab', F(ab')₂ or F(v) portions of whole antibody molecules.

As suggested earlier, the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to a receptor recognition factor/protein, such as an anti-recognition factor antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb. In addition, it is preferable for the anti-recognition factor antibody molecules used herein be in the form of Fab, Fab', F(ab')₂ or F(v) portions or whole antibody molecules. As previously discussed, patients capable of benefiting from this method include those suffering from cancer, a pre-cancerous lesion, a viral infection or other like pathological derangement. Methods for isolating the recognition factor and inducing anti-recognition factor antibodies and for determining and optimizing the ability of anti-recognition factor antibodies to assist in the examination of the target cells are all well-known in the art.

Methods for producing polyclonal anti-polypeptide antibodies are well-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et al. A monoclonal antibody, typically containing Fab and/or F(ab')₂ portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodies--A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a recognition factor-binding portion thereof, or recognition factor, or an origin-specific DNA-binding portion thereof.

Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present recognition factor and their ability to inhibit specified transcriptional activity in target cells.

A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.

The antibody-containing medium is then collected. The antibody molecules can then be futher isolated by well-known techniques.

Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-recognition factor antibodies are also well-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983). Typically, the present recognition factor or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen in the before described procedure for producing anti-recognition factor monoclonal antibodies. The hybridomas are screened for the ability to produce an antibody that immunoreacts with the recognition factor peptide analog and the present recognition factor.

The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a receptor recognition factor, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient. In a preferred embodiment, the composition comprises an antigen capable of modulating the specific binding of the present recognition factor within a target cell.

The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of recognition factor binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.

The therapeutic compositions may further include an effective amount of the factor/factor synthesis promoter antagonist or analog thereof, and one or more of the following active ingredients: an antibiotic, a steroid. Exemplary formulations are given below:

    ______________________________________                                         Formulations                                                                   Ingredient              mg/ml                                                  ______________________________________                                         Intravenous Formulation I                                                      cefotaxime              250.0                                                  receptor recognition factor                                                                             10.0                                                  dextrose USP             45.0                                                  sodium bisulfite USP     3.2                                                   edetate disodium USP     0.1                                                   water for injection q.s.a.d.                                                                            1.0 ml                                                Intravenous Formulation II                                                     ampicillin              250.0                                                  receptor recognition factor                                                                             10.0                                                  sodium bisulfite USP     3.2                                                   disodium edetate USP     0.1                                                   water for injection q.s.a.d.                                                                            1.0 ml                                                Intravenous Formulation III                                                    gentamicin (charged as sulfate)                                                                         40.0                                                  receptor recognition factor                                                                             10.0                                                  sodium bisulfite USP     3.2                                                   disodium edetate USP     0.1                                                   water for injection q.s.a.d.                                                                            1.0 ml                                                Intravenous Formulation IV                                                     recognition factor       10.0                                                  dextrose USP             45.0                                                  sodium bisulfite USP     3.2                                                   edetate disodium USP     0.1                                                   water for injection q.s.a.d.                                                                            1.0 ml                                                Intravenous Formulation V                                                      recognition factor antagonist                                                                           5.0                                                   sodium bisulfite USP     3.2                                                   disodium edetate USP     0.1                                                   water for injection q.s.a.d.                                                                            1.0 ml                                                ______________________________________                                    

As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" mean microgram, "mg" means milligram, "ul" or "μl" mean microliter, "ml" means milliliter, "l" means liter.

Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and Synthetic DNA sequences. Suitable vectors include derivatives of SV40 and nown bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, and other phage DNA, e.g., M13 and Filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAS, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Any of a wide variety of expression control sequences--sequences that control the expression of a DNA sequence operatively linked to it--may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast α-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system.

However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

It is further intended that receptor recognition factor analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of receptor recognition factor material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of receptor recognition factor coding sequences. Analogs exhibiting "receptor recognition factor activity" such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding receptor recognition factor can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the receptor recognition factor amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes which will express receptor recognition factor analogs or "muteins". Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native receptor recognition factor genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science, 244:182-188 (April 1989). This method may be used to create analogs with unnatural amino acids.

The present invention extends to the preparation of antisense nucleoddes and ribozymes that may be used to interfere with the expression of the receptor recognition proteins at the translational level. This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. (See Weintraub, 1990; Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into receptor recognition factor-producing cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988.). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-type and "hammerhead"-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead"-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.

The DNA sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave mRNAs for receptor recognition factor proteins and their ligands.

The present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as the earlier referenced polypeptide ligands, by reference to their ability to elicit the activities which are mediated by the present receptor recognition factor. As mentioned earlier, the receptor recognition factor can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular transcriptional activity in suspect target cells.

As described in detail above, antibody(ies) to the receptor recognition factor can be produced and isolated by standard methods including the well known hybridoma techniques. For convenience, the antibody(ies) to the receptor recognition factor will be referred to herein as Ab₁ and antibody(ies) raised in another species as Ab₂.

The presence of receptor recognition factor in cells can be ascertained by the usual immunological procedures applicable to such determinations. A number of useful procedures are known. Three such procedures which are especially useful utilize either the receptor recognition factor labeled with a detectable label, antibody Ab₁ labeled with a detectable label, or antibody Ab₂ labeled with a detectable label. The procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "RRF" stands for the receptor recognition factor:

A. RRF*+Ab₁ =RRF*Ab₁

B. RRF+Ab*=RRFAb₁ *

C. RRF+Ab₁ +Ab₂ *=RRFAb₁ Ab₂ *

The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure, Procedure A, is described in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the "sandwich" procedure, is described in U.S. Pat. Nos. RE 31,006 and 4,016,043. Still other procedures are known such as the "double antibody", or "DASP" procedure.

In each instance, the receptor recognition factor forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label. The fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ is that it will react with Ab₁. This is because Ab₁ raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab₂. For example, Ab₂ may be raised in goats using rabbit antibodies as antigens. Ab₁ therefore would be anti-rabbit antibody raised in goats. For purposes of this description and claims, Ab₁ will be referred to as a primary or anti-receptor recognition factor antibody, and Ab₂ will be referred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.

A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine and auramine. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

The receptor recognition factor or its binding partner(s) can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, and ¹⁸⁶ Re.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

A particular assay system developed and utilized in accordance with the present invention, is known as a receptor assay. In a receptor assay, the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.

Accordingly, a purified quantity of the receptor recognition factor may be radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined receptor recognition factor, and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of <5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.

An assay useful and contemplated in accordance with the present invention is known as a "cis/trans" assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex. Thus, for example, if it is desired to evaluate a compound as a ligand for a particular receptor, one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted. If the compound under test is an agonist for the receptor, the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene. The resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands. The foregoing protocol is described in detail in U.S. Pat. No. 4,981,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.

In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined transcriptional activity or predetermined transcriptional activity capability in suspected target cells. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled receptor recognition factor or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive", "sandwich", "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of the presence or capability of cells for predetermined transcriptional activity, comprising:

(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present receptor recognition factor or a specific binding partner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of the receptor recognition factor as described above (or a binding partner) generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural such end products, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. "competitive", "sandwich", "double antibody", etc.), and comprises:

(a) a labeled component which has been obtained by coupling the receptor recognition factor to a detectable label;

(b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeled component (a);

(iii) a ligand capable of binding with at least one of the component(s) to be determined; and

(iv) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the receptor recognition factor and a specific binding partner thereto.

In accordance with the above, an assay system for screening potential drugs effective to modulate the activity of the receptor recognition factor may be prepared. The receptor recognition factor may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the transcriptional activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known receptor recognition factor.

PRELIMINARY CONSIDERATIONS

As mentioned earlier, the observation and conclusion underlying the present invention were crystallized from a consideration of the results of certain investigations with particular stimuli. Particularly, the present disclosure is illustrated by the results of work on protein factors that govern transcriptional control of IFNα-stimulated genes, as well as more recent data on the regulation of transcription of genes stimulated by IFNγ. The following is a brief discussion of the role that IFN is believed to play in the stimulation of transcription taken from Darnell et al. THE NEW BIOLOGIST, 2(10), (1990).

Activation of genes by IFNα occurs within minutes of exposure of cells to this factor (Larner et al., 1984, 1986) and is strictly dependent on the IFNα binding to its receptor, a 49-kD plasma membrane polypepdde (Uze et al., 1990). However, changes in intracellular second messenger concentrations secondary to the use of phorbol esters, calcium ionophores, or cyclic nucleotide analogs neither triggers nor blocks IFNα-dependent gene activation (Larrer et al., 1984; Lew et al., 1989). No other polypeptide, even IFNγ, induces the set of interferon-stimulated genes (ISGS) specifically induced by IFNα. In addition, it has been found that IFNγ-dependent transcriptional stimulation of at least one gene in HeLa cells and in fibroblasts is also strictly dependent on receptor-ligand interaction and is not activated by induced changes in second messengers (Decker et al., 1989; Lew et al., 1989). These highly specific receptor-ligand interactions, as well as the precise transcriptional response, require the intracellular recognition of receptor occupation and the communication to the nucleus to be equally specific.

The activation of ISGs by IFNα is carried out by transcriptional factor ISGF-3, or interferon stimulated gene factor 3. This factor is activated promptly after IFNα treatment without protein synthesis, as is transcription itself (Larner et al., 1986;

Levy et al., 1988; Levy et al., 1989). ISGF-3 binds to the ISRE, the interferon-stimulated response element, in DNA of the response genes (Reich et al., 1987; Levy et al., 1988), and this binding is affected by all of an extensive set of mutations that also affects the transcriptional function of the ISRE (Kessler et al., 1988a). Partially purified ISGF-3 containing no other DNA-binding components can stimulate ISRE-dependent in vitro transcription (Fu et al., 1990). IFN-dependent stimulation of ISGs occurs in a cycle, reaching a peak of 2 hours and declining promptly thereafter (Larner et al., 1986). ISGF-3 follows the same cycle (Levy et al., 1988, 1989). Finally, the presence or absence or ISGF3 in a variety of IFN-sensitive and IFN-resistant cells correlates with the transcription of ISGs in these cells (Kessler et al., 1988b).

ISGF-3 is composed of two subfractions, ISGF-3α and ISGF-3-γ, that are found in the cytoplasm before IFN binds to its receptor (Levy et al., 1989). When cells are treated with IFNα, ISGF-3 can be detected in the cytoplasm within a minute, that is, some 3 to 4 minutes before any ISGF-3 is found in the nucleus (Levy et al., 1989). The cytoplasmic component ISGF-3γ can be increased in HeLa cells by pretreatment with IFNγ, but IFNγ does not by itself activate transcription of ISGs nor raise the concentration of the complete factor, ISGF-3 (Levy et al., 1990). The cytoplasmic localization of the proteins that interact to constitute ISGF-3 was proved by two kinds of experiments. When cytoplasm of IFNγ-treated cells that lack ISGF-3 was mixed with cytoplasm of IFNα-treated cells, large amounts of ISGF-3 were formed (Levy et al., 1989). (It was this experiment that indicated the existence of an ISGF-3γ component and an ISGF-3α component of ISGF-3).

In addition, Dale et al. (1989) showed that enucleated cells could respond to IFNα by forming a DNA-binding protein that is probably the same as ISGF-3.

The ISGF-3γ component is a 48-kD protein that specifically recognizes the ISRE (Kessler et al., 1990; Fu et al., 1990). Three other proteins, presumably constituting the ISGF-3α component, were found in an ISGF-3 DNA complex (Fu et al., 1990). The entirety of roles of, or the relationships among these three proteins are not yet known, but it is clear that ISGF-3 is a multimeric protein complex. Since the binding of IFNα to the cell surface converts ISGF-3α from an inactive to an active status within a minute, at least one of the proteins constituting ISGF-3α must be affected promptly, perhaps by a direct interaction with the IFNα receptor.

The details of how the ISGF-3γ component and the three other proteins are activated by cytoplasmic events and then enter the nucleus to bind the ISRE and increase transcription are not entirely known. Further studies of the individual proteins, for example, with antibodies, are presented herein. For example, it is clear that, within 10 minutes of IFNα treatment, there is more ISGF-3 in the nucleus than in the cytoplasm and that the complete factor has a much higher affinity for the ISRE than the 48-kD ISGF-3γ component by itself (Kessler et al., 1990).

In summary, the attachment of interferon-α (IFN-α) to its specific cell surface receptor activates the transcription of a limited set of genes, termed ISGs for "interferon stimulated genes" [Larner et al., PROC. NATL. ACAD. SCI. USA, 81 (1984); Larner et al., J. BIOL. CHEM., 261 (1986); Friedman et al., CELL, 38 (1984)]). The observation that agents that affect second messenger levels do not activate transcription of these genes, led to the proposal that protein:protein interactions in the cytoplasm beginning at the IFN receptor might act directly in transmitting to the nucleus the signal generated by receptor occupation [Levy et al., NEW BIOLOGIST, 2 (1991)].

To test this hypothesis, the present applicants began experiments in the nucleus at the activated genes. Initially, the ISRE and ISGF-3 were discovered [Levy et al., GENES & DEV., 2 (1988)].

Partial purification of ISGF-3 followed by recovery of the purified proteins from a specific DNA-protein complex revealed that the complete complex was made up of four proteins [Fu et al., PROC. NATL. ACAD. SCI. USA, 87 (1990); Kessler et al., GENES & DEV., 4 (1990)]. A 48 kD protein termed ISGF-3γ, because pre-treatment of HeLa cells with IFN-γ increased its presence, binds DNA weakly on its own [Ibid.; and Levy et al., THE EMBO. J., 9 (1990)]. In combination with the IFN-α activated proteins, termed collectively the ISGF-3α proteins, the ISGF-3γ forms a complex that binds the ISRE with a 50-fold higher affinity [Kessler et al., GENES & DEV, 4 (1990)]. The ISGF-3α proteins comprise a set of polypeptides of 113, 91 and 84 kD. All of the ISGF-3 components initially reside in the cell cytoplasm [Levy et al., GENES & DEV., 3 (1989); Dale et al., PROC. NATL. ACAD. SCI. USA, 86 (1989)]. However after only about five minutes of IFN-α treatment the active complex is found in the cell nucleus, thus confirming these proteins as a possible specific link from an occupied receptor to a limited set of genes [Levy et al., GENES & DEV., 3 (1989)].

In accordance with the present invention, specific proteins comprising receptor recognition factors have been isolated and sequenced. These proteins, their fragments, antibodies and other constructs and uses thereof, are contemplated and presented herein. To understand the mechanism of cytoplasmic activation of the ISGF-3α proteins as well as their transport to the nucleus and interaction with ISGF-3γ, this factor has been purified in sufficient quantity to obtain peptide sequence from each protein. Degenerate deoxyoligonucleotides that would encode the peptides were constructed and used in a combination of cDNA library screening and PCR amplification of cDNA products copied from mRNA to identify cDNA clones encoding each of the four proteins. What follows in the examples presented herein a description of the final protein preparations that allowed the cloning of cDNAs encoding all the proteins, and the primary sequence of the 113 kD protein arising from a first gene, and the primary sequences of the 91 and 84 kD proteins which appear to arise from two differently processed RNA products from another gene. Antisera against portions of the 84 and 91 kD proteins have also been prepared and bind specifically to the ISGF-3 DNA binding factor (detected by the electrophoretic mobility shift assay with cell extracts) indicating that these cloned proteins are indeed part of ISGF-3. The availability of the cDNA and the proteins they encode provides the necessary material to understand how the liganded IFN-α receptor causes immediate cytoplasmic activation of the ISGF-3 protein complex, as well as to understand the mechanisms of action of the receptor recognition factors contemplated herein. The cloning of each of ISGF3-α proteins, and the evaluation and confirmation of the particular role played by the 91 kD protein as a messenger and DNA binding protein in response to IFN-γ activation, including the development and testing of antibodies to the receptor recognition factors of the present invention, are all presented in the examples that follow below.

EXAMPLE 1

To purify relatively large amounts of ISGF-3, HeLa cell nuclear extracts were prepared from cells treated overnight (16-18 h) with 0.5 ng/ml of IFN-γ and 45 min. with IFN-α (500 μ/ml). The steps used in the large scale purification were modified slightly from those described earlier in the identification of the four ISGF-3 proteins.

Accordingly, nuclear extracts were made from superinduced HeLa cells [Lvy et al., THE EMBO. J., 9 (1990)] and chromatographed as previously described [Fu et al., PROC. NATL. ACAD. SCI. USA, 87 (1990)] on: phosphocellulose P-11, heparin agarose (Sigma); DNA cellulose (Boehringer Mannheim; flow through was collected after the material was adjusted to 0.28M KCl and 0.5% NP-40); two successive rounds of ISRE oligo affinity column (1.8 ml column, eluted with a linear gradient of 0.05 to 1.0M KCl); a point mutant ISRE oligonucleotide affinity column (flow through was collected after the material was adjusted to 0.28M KCl); and a final round on the ISRE oligonucleotide column (material was eluted in a linear 0.05 to 1.0M NaCl gradient adjusted to 0.05% NP-40). Column fractions containing ISGF-3 were subsequently examined for purity by SDS PAGE/silver staining and pooled appropriately. The pooled fractions were concentrated by a centricon-10 (Amicon). The pools of fractions from preparations 1 and 2 were combined and run on a 10 cm wide, 1.5 mm thick 7.5% SDS polyacrylamide gel. The proteins were electroblotted to nitrocellulose for 12 hrs at 20 volts in 12.5% MeOH, 25 mM Tris, 190 mM glycine. The membrane was stained with 0.1% Ponceau Red (in 1% acetic acid) and the bands of 113 kD, 91 kD, 84 kD, and 48 kD excised and subjected to peptide analysis after tryptic digestion [Wedrychowski et al., J. BIOL. CHEM., 265 (1990); Aebersold et al., PROC. NATL. ACAD. SCI. USA, 84 (1987)]. The resulting peptide sequences for the 91 kD and 84 kD proteins are indicated in FIG. 6. Degenerate oligonucleotides were designed based on the peptide sequences t19, t13b and t27: (Forward and Reverse complements are denoted by F and R:

    19F AACGTIGACCAATTNAACATG                                                                             (SEQ ID NO:14)                                                T     T  GC    T                                                                 T                                                                      13bR GTCGATGTTNGGGTANAG                                                                               (SEQ ID NO:15)                                               A  A   A   A     A                                                        27R GTACAAITCAACCAGNGCAA                                                                              (SEQ ID NO:16)                                                 T    TG  T     T                                                   

The final ISRE oligonucleotide affinity selection yielded material with the SDS polyacrylarnide gel electrophoretic pattern shown in FIG. 4 (left). This gel represented about 1.5% of the available material purified from over 200 L of appropriately treated HeLa cells. While 113, 91, 84 and 48 kD bands were clearly prominent in the final purified preparation (see FIG. 4, right panel), there were also two prominent contaninants of about 118 and 70 kD and a few of other contaminants in lower amounts. [Amino acid sequence data have shown that the contaminants of 86 kD and 70 kD are the KU antigen, a widely-distributed protein that binds DNA termini. However in the specific ISGF-3: ISRE complex there is no KU antigen and therefore it has been assigned no role in IFN-dependent transcriptional stimulation, [Wedrychowski et al., J. BIOL. CHEM., 265 (1990)].

Since the mobility of the 113, 91, 84, and 48 kD proteins could be accurately marked by comparison with the partially purified proteins characterized in previous experiments [Fu et al., PROC. NATL. ACAD. SCI. USA, 87 (1990)], further purification was not attempted at this stage. The total purified sample from 200 L of HeLa cells was loaded onto one gel, subjected to electrophoresis, transferred to nitrocellulose and stained with Ponceau red. The 113, 84, 91, and 48 kD protein bands were separately excised and subjected to peptide analysis as described [Aebersold et al., PROC NATL. ACAD. SCI. USA, 84 (1987)]. Released peptides were collected, separated by HPLC and analyzed for sequence content by automated Edman degradation analysis.

Accordingly, the use of the peptide sequence data for three of four peptides from the 91 kD protein and a single peptide derived from the 84 kD protein is described herein. The peptide sequence and the oligonucleotides constructed from them are given in the legend to FIGS. 4 or 6. When oligonucleotides 19F and 13bR were used to prime synthesis from a HeLa cell cDNA library, a PCR product of 475 bp was generated. When this product was cloned and sequenced it encoded the 13a peptide internally. Oligonucleotide 27R derived from the only available 84 kD peptide sequence was used in an anchored PCR procedure amplifying a 405 bp segment of DNA. This 405 bp amplified sequence was identical to an already sequenced region of the 91 kD protein. It was then realized that the peptide t27 sequence was contained within peptide t19 and that the 91 and 84 kD proteins must be related (see FIGS. 5 & 7). Oligonucleotides 19F and 13a were also used to select candidate cDNA clones from a cDNA library made from mRNA prepared after 16 hr. of IFN-γ and 45 min. of IFN-α treatment.

Of the numerous cDNA clones that hybridized these oligonucleotides and also the cloned PCR products, one cDNA clone, E4, contained the largest open reading frame flanked by inframe stop codons. Sequence of peptides t19, t13a, and t13b were contained in this 2217 bp ORF (see FIG. 6) which was sufficient to encode a protein of 739 amino acids (calculated molecular weight of 86 kD). The codon for the indicated initial methionine was preceded by three in frame stop codons. This coding capacity has been confirmed by translating in vitro an RNA copy of the E4 clone yielding product of nominal size of 86 kD, somewhat shorter than the in vitro purified 91 kD protein discussed earlier (data not shown). Perhaps this result indicates post-translational modification of the protein in the cell.

A second class of clones was also identified (see FIG. 5). E3, the prototype of this class was identical to E4 from the 5' end to bp 2286 (aa 701) at which point the sequences diverged completely. Both cDNAs terminated with a poly(A) tail. Primer extension analysis suggested another ˜150 bp were missing from the 5' end of both mRNAs. DNA probes were made from the clones representing both common and unique sequences for use in Northern blot analyses. The preparation of the probes is as follows: 20 mg of cytoplasmic RNA (0.5% NP-40 lysate) of IFN-α treated (6 h) HeLa RNA was fractionated in a 1% agarose, 6% formaldehyde gel (in 20 mM MOPS, 5mM NaAc, 1 mM EDTA, pH 7.0) for 4.5 h at 125 volts. The RNA was transferred in 20×SSC to Hybond-N (Amersham), UV crosslinked and hybridized with 1×106 cpm/ml of the indicated probes (1.5×10⁸ cpm/mg).

Probes from regions common to E3 and E4 hybridized to two RNA species of approximately 3.1 KB and 4.4 KB. Several probes derived from the 3' non-coding end of E4, which were unique to E4, hybridized only the larger RNA species. A labeled DNA probe from the unique 3' non-coding end of E3 hybridized only the smaller RNA species.

Review of the sequence at the site of 3' discontinuity between E3 and E4 suggested that the shorter mRNA results from choice of a different poly(A) site and 3' exon that begins at bp 2286 (the calculated molecular weight from the E3. The last two nucleotides before the change are GT followed by GT in E3 in line with the consensus nucleotides at an exon-intron junction. Since the ORF of E4 extends to bp 2401 it encodes a protein that is 38 amino acids longer than the one encoded by E3, but is otherwise identical (ORF is 82 kD).

Since there is no direct assay for the activity of the 91 or 84 kD protein, an independent method was needed to determine whether the cDNA clones we had isolated did indeed encode proteins that are part of ISGF-3. For this purpose antibodies were initially raised against the sequence from amino acid 597 to amino acid 703 (see FIG. 6) by expressing this peptide in the pGEX-3X vector (15) as a bacterial fusion protein. This antiserum (a42) specifically recognized the 91 kD and 84 kD proteins in both crude extracts and purified ISGF-3 (see FIG. 7a). More importantly this antiserum specifically affected the ISGF-3 band in a mobility shift assay using the labeled ISRE oligonucleotide (see FIG. 7b) confirming that the isolated 91 kD and 84 kD cDNA clones (E4 and E3) represent a component of ISGF-3. Additional antisera were raised against the amino terminus and carboxy terminus of the protein encoded by E4. The amino terminal 59 amino acids that are common to both proteins and the unique carboxy terminal 34 amino acids encoded only by the larger mRNA were expressed as fusion proteins in pGEX-3X for immunization of rabbits. Western blot analysis with highly purified ISGF-3 demonstrated that the amino terminal antibody (a55) recognized both the 91 kD and 84 kD proteins as expected. However, the other antibody (a57) recognized only the 91 kD protein confirming our assumption that the larger mRNA (4.4 KB) and larger cDNA encodes the 91 kD protein while the shorter mRNA (3.1 KB) and cDNA encodes the 84 kD protein (see FIG. 7a).

EXAMPLE 2

In this example, the cloning of the 113 kD protein that comprises one of the three ISGF-3α components is disclosed.

From SDS gels of highly purified ISGF-3, the 113 kD band was identified, excised and subjected to cleavage and peptide sequence analysis [Aebersold et al., PROC. NATL. ACAD. SCI. USA, 87 (1987)]. Five peptide sequences (A-E) were obtained (FIG. 8A). Degenerate oligonucleotide probes were designed according to these peptides which then were radiolabeled to search a human cDNA library for clones that might encode the 113 kD protein. Eighteen positive cDNA clones were recovered from 2.5×10⁵ phage plaques with the probe derived from peptide E (FIG. 8A, and the legend). Two of them were completely sequenced. Clone f11 contained a 3.2 KB cDNA, and clone ka31 a 2.6 KB cDNA that overlapped about 2 KB but which had a further extended 5' end in which a candidate AUG initiation codon was found associated with a well-conserved Kozak sequence [Kozak, NUCLEIC ACIDS RES., 12 (1984)].

In addition to the phage cDNA clones, a PCR product made between oligonucleotides that encoded peptide D and E also yielded a 474 NT fragment that when sequenced was identical with the cDNA clone in this region. A combination of these clones f11 and ka31 revealed an open reading frame capable of encoding a polypeptide of 851 amino acids (FIG. 8A). These two clones were joined within their overlapping region and RNA transcribed from this recombinant clone was translated in vitro yielding a polypeptide that migrated in an SDS gel with a nominal molecular weight of 105 kD (FIG. 9A). An appropriate clone encoding the 91 kD protein was also transcribed and the RNA translated in the same experiment. Since both the apparently complete cDNA clones for the 113 kD protein and the 91 kD protein produce RNAs that when translated into proteins migrate somewhat faster than the proteins purified as ISGF-3 components, it is possible that the proteins undergo post-translational modification in the cell causing them to be slightly retarded during electrophoresis. When a 660 bp cDNA encoding the most 3' end of the 113 kD protein was used in a Northern analysis, a single 4.8 kB mRNA species was observed (FIG. 9B).

No independent assay is known for the activity of the 113 kD (or indeed any of the ISGF-3α proteins,) but it is known that the protein is part of a DNA binding complex that can be detected by an electrophoretic mobility shift assay [Fu et al., PROC. NATL. ACAD. SCI. USA, 87 (1990)]. Antibodies to DNA binding proteins are known to affect the formation or migration of such complexes. Therefore antiserum to a polypeptide segment (amino acid residues 323 to 527) fused with bacterial glutathione synthetase [Smith et al., PROC. NATL. ACAD. SCI. USA, 83 (1986)] was raised in rabbits to determine the reactivity of the ISGF-3 proteins with the antibody. A Western blot analysis showed that the antiserum reacted predominantly with a 113 kD protein both in the ISGF3 fraction purified by specific DNA affinity chromatography (Lane 1) and in crude cell extract (Lane 2, FIG. 10A). The weak reactivity to lower protein bands was possibly due to 113 kD protein degradation. Most importantly, the antiserum specifically removed almost all of the gel-shift complex leaving some of the oligonucleotide probe in "shifted-shift" complexes which were specifically competed away with a 50 fold molar excess of the oligonucleotide binding site (the ISRE, ref. 2) for ISGF3 (FIG. 10B). Notably, this antiserum had no effect on the faster migrating shift band produced by ISGF3-γ component alone (FIG. 10B). Thus it appeared that the antiserum to the 113 kD fusion product does indeed react with another protein that is part of the complete ISGF-3 complex.

A detailed sequence comparison between the 113 and 91 sequences followed (FIG. 8B): while the nucleotide sequence showed only a distant relationship between the two proteins, there were long stretches of amino acid identity. These conserved regions were scattered throughout almost the entire 715 amino acid length encoded by the 91/84 clone. It was particularly striking that the regions corresponding to amino acids 1 to 48 and 317 to 353 and 654 to 678 in the 113 sequence were 60% to 70% identical to corresponding regions of the 91 kD sequence. Thus the genes encoding the 113 and 84/91 proteins are closely related but not identical.

Through examination for possible consensus sequences that might reveal sub-domain structures in the 113 kD or 84/91 kD sequence, it was found that both proteins contained regions whose sequence might form a coil structure with heptad leucine repeats. This occurred between amino acid 210 and 245 in the 113 kD protein and between 209 and 237 in the 84/91 protein. In both the 113 kD and the 91/84 kD sequences, 4 out of 5 possible heptad repeats were leucine and one was valine. Domains of this type might provide a protein surface that encourages homo-or heterotypic protein interactions which have been observed in several other transcription factors [Vinson et al., SCIENCE, 246 (1989)]. An extended acidic domain was located at the carboxyl terminal of the 113 kD protein but not in 91 kD protein (FIG. 8A), possibly implicating the 113 kD protein in gene activation [Hope et al., Ma et al., CELL, 48 (1987)].

DISCUSSION

When compared at moderate or high stringency to the Genbank and EMBL data bases, there were no sequences like 113 or the 84/91 sequence. Preliminary PCR experiments however indicate that there are other family members with different sequences recoverable from a human cell cDNA library (Qureshi and Darnell unpublished). Thus, it appears that the 113 and 84/91 sequences may represent the first two members to be cloned of a larger family of proteins. We would hypothesize that the 113 kD and 84/91 kD proteins may act as signal transducers, somehow interacting with the internal domain of a liganded IFNα receptor or its associated protein and further that a family of waiting cytoplasmic proteins exist whose purpose is to be specific signal transducers when different receptors are occupied. Many experiments lie ahead before this general hypothesis can be crucially tested. Recent experiments have indicated that inhibitors of protein kinases can prevent ISGF-3 complex formulation Reich et al., PROC. NATL. ACAD. SCI. USA, 87 (1990); Kessler et al., J. BIOL. CHEM., 266 (1991)].

However, neither the IFNα or IFNγ receptors that have so far been cloned have intrinsic kinase activity [Uze et al., CELL, 60 (1990); Aguet et al., CELL, 55 (1988)]. We would speculate that either a second receptor chain with kinase activity or a separate kinase bound to a liganded receptor could be a part of a complex that would convey signals to the ISGF-3α proteins at the inner surface of the plasma membrane.

From the above, it has been concluded that accurate peptide sequence from ISGF-3 protein components have been determined, leading to correct identification of cDNA clones encoding the 113, 91 and 84 kD components of ISGF-3 . Since staurosporine, a broadly effective kinase inhibitor blocks IFN-α induction of transcription and of ISGF-3 formation Reich et al., PROC. NATL. ACAD. SCI. USA, 87 (1990); Kessler et al., J. BIOL. CHEM., 266 (1991)] it seems possible that the ISGF-3α proteins are direct cytoplasmic substrates of a liganded receptor-associated kinase. The antiserum against these proteins should prove invaluable in identifying the state of the ISGF-3α proteins before and after IFN treatment and will allow the direct exploration of the biochemistry of signal transduction from the IFN receptor.

EXAMPLE 3

As mentioned earlier, the observation and conclusion underlying the present invention were crystallized from a consideration of the results of certain investigations with particular stimuli. Particularly, the present disclosure is illustrated by the results of work on protein factors that govern transcriptional control of IFNα-stimulated genes, as well as more recent data on the regulation of transcription of genes stimulated by IFNγ.

For example, there is evidence that the 91 kD protein is the tyrosine kinase target when IFNγ is the ligand. Thus two different ligands acting through two different receptors both use these family members. With only a modest number of family members and combinatorial use in response to different ligands, this family of proteins becomes an even more likely possibility to represent a general link between ligandoccupied receptors and transcriptional control of specific genes in the nucleus.

Further study of the 113, 91 and 84 kD proteins of the present invention has revealed that they are phosphorylated in response to treatment of cells with IFNα (FIG. 11). Moreover, when the phosphoamino acid is determined in the newly phosphorylated protein the amino acid has been found to be tyrosine (FIG. 12). This phosphorylation has been observed to disappear after several hours, indicating action of a phosphatase of the 113, 91 and 84 kD proteins to stop transcription. These results show that IFN dependent transcription very likely demands this particular phosphorylation and a cycle of interferon-dependent phosphorylation-dephosphorylation is responsible for controlling transcription.

It is proposed that other members of the 113-91 protein family will be identified as phosphorylation targets in response to other ligands. If as is believed, the tyrosine phosphorylation site on proteins in this family is conserved, one can then easily determine which family members are activated (phosphorylated), and likewise the particular extracellular polypeptide ligand to which that family member is responding. The modifications of these proteins (phosphorylation and dephosphorylation) enables the preparation and use of assays for determining the effectiveness of pharmaceuticals in potentiating or preventing intracellular responses to various polypeptides, and such assays are accordingly contemplated within the scope of the present invention.

EXAMPLE 4

Identification of Murine 91 kD Protein

A fragment of the gene encoding the human 91 kD protein was used to screen a murine thymus and spleen cDNA library for homologous proteins. The screening assay yielded a highly homologous gene encoding a murine polypeptide that is greater than 95% homologous to the human 91 kD protein. The nucleic acid and deduced amino acid sequence of the murine 91 kD protein are shown in FIGS. 13A-13C, and SEQ ID NO:7 (nucleotide sequence) and SEQ ID NO:8 (amino acid sequence).

EXAMPLE 5

Additional Members of The 113-91 Protein Family

Using a 300 nuclide fragment amplified by PCR from the SH2 region of the murine 91 kD protein gene, murine genes encoding two additional members of the 113-91 family of receptor recognition factor proteins were isolated from a murine splenic/thymic cDNA library according to the method of Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, 2nd. ed., Cold Spring Harbor Press: Cold Spring Harbor, N.Y.) constructed in the ZAP vector. Hybridization was carried out at 42° C. and washed at 42° C. before the first exposure (Church and Gilbert, 1984, Proc. Natl. Acad. Sci. USA 81:1991-95). Then the filters were washed in 2×SSC, 0.1% SDS at 65° C. for a second exposure. Stat1 clones survived the 65° C. washing, whereas Stat3 and Stat4 clones were identified as plaques that lost signals at 65° C. The plaques were purified and subcloned according to Stratagene commercial protocols.

This probe was chosen to screen for other STAT family members because, while Stat1 and Stat2 SH2 domains are quite similar over the entire 100 to 120 amino acid region, only the amino terminal half of the STAT SH2 domains strongly resemble the SH2 regions found in other proteins.

The two genes have been cloned into plasmids 13sf1 and 19sf6. The nucleotide sequence, and deduced amino acid sequence, for the 13sf1 and 19sf6 genes are shown in FIGS. 14 and 15, respectively. These proteins are alternatively termed Stat4 and Stat3, respectively.

Comparison with the sequence of Stat91 (Stat1) and Stat113 (Stat2) shows several highly conserved regions, including the putative SH3 and SH2 domains. The conserved amino acid stretches likely point to conserved domains that enable these proteins to carry out transcription activation functions. Stat3, like Stat1 (Stat91), is widely expressed, while Stat4 expression is limited to the testes, thymus, and spleen. Stat3 has been found to be activated as a DNA binding protein through phosphorylation on tyrosine in cells treated with EGF or IL-6, but not after IFN-γ treatment.

Both the 13sf1 and 19sf6 genes share a significant homology with the genes encoding the human and murine 91 kD protein. There is corresponding homology between the deduced amino acid sequences of the 13sf1 and 19sf6 proteins and the amino acid sequences of the human and murine 91 kD proteins, although not the greater than 95% amino acid homology that is found between the murine and human 91 kD proteins. Thus, though clearly of the same family as the 91 kD protein, the 13sf1 and 19sf6 genes encode distinct proteins.

The chromosomal locations of the murine STAT proteins (1-4) have been determined: Stat1 and Stat4 are located in the centromeric region of mouse chromosome 1 (corresponding to human 2q 32-34q); the two other genes are on other chromosomes.

Southern analysis using probes derived from 13sf1 and 19sf6 on human genomic libraries have established that genes corresponding to the murine 13sf1 and 19sf6 genes are found in humans.

Tissue distribution of mRNA expression of these genes was evaluated by Northern hybridization analysis. The results of this distribution analysis are shown in the following Table.

                  TABLE                                                            ______________________________________                                         DISTRIBUTION OF mRNA EXPRESSION OF                                             13sfl, 19sf6, 91 kD PROTEINS                                                   ORGAN          13sfl      19sf6    91 KD                                       ______________________________________                                         BRAIN          -          +        -                                           HEART          -          +++      -                                           KIDNEY         -          -        -                                           LIVER          -          +        +                                           LUNG           -          -        -                                           SPLEEN         +          +        ++++                                        TESTIS         ++++       ++       N.A.                                        THYMUS         ++         ++       +++                                         EMBRYO (16d)   not found  found    found                                       ______________________________________                                    

Northern analysis demonstrates that there is variation in the tissue distribution of expression of the mRNAs encoded by these genes. The variation and tissue distribution indicates that the specific genes encode proteins that are responsive to different factors, as would be expected in accordance with the present invention. The actual ligand, the binding of which induces phosphorylation of the newly discovered factors, will be readily determinable based on the tissue distribution evidence described above.

To determine whether the Stat3 and Stat4 proteins were present in cells, protein blots were carried out with antisera against each protein. The antisera were obtained by subcloning amino acids 688 to 727 of Stat3 and 678 to 743 of Stat4 to pGEX1λt (Pharmacia) by PCR with oligonucleotides based on the boundary sequence plus restriction sites (BamHi at the 5' end and EcoRI at the 3' end), allowing for in-frame fusion with GST. One milligram of each antigen was used for the immunization and three booster injections were given 4 weeks apart. Anti-Stat3 and anti-Stat4 sera were used 1:1000 in Western blots using standard protocols. To avoid cross reactivity of the antisera, antibodies were raised against the C-terminal of Stat3 and Stat4, the less homologous region of the protein.

These proteins were unambiguously found in several tissues where the mRNA was known to be present. Protein expression was checked in several cell lines as well. A protein of 89 kD reactive with Stat4 antiserum was expressed in 70Z cells, a preB cell line, but not in many other cell lines. Stat3 was highly expressed, predominantly as a 97 kD protein, in 70Z, HT2 (a mouse helper T cell clone), and U937 (a macrophage-derived cell).

To prove that the full length functional cDNA clones of Stat3 and Stat4 were obtained, the open reading frames of each cDNA was independently (i.e., separately) cloned into the Rc/CMV expression vector (Invitrogen) downstream of a CMV promoter. The resulting plasmids were transfected into COS1 cells and proteins were extracted 60 hrs post-transfection and examined by Western blot after electrophoresis. Untransfected COS1 cells expressed a low level of 97 kD Stat3 protein but did not express a detectable level of Stat4. Upon transfection of the Stat3-expressing plasmid, the 97 kD Stat3 was increased at least 10-fold. And 89 kD protein antigenically related to Stat3, found as a minor band in most cell line extracts, was also increased post-transfection. This protein therefore appears to represent another form of Stat3 protein, or an antigenically similar protein whose synthesis is stimulated by Stat3. Transfection with Stat4 led to the expression of a 89 kD reactive band indistinguishable in size form the p89 Stat4 found in 70Z cell extracts.

DISCUSSION

As mentioned earlier, the observation and conclusion underlying the present invention were crystallized from a consideration of the results of certain investigations with particular stimuli. Particularly, the present disclosure is illustrated by the results of work on protein factors that govern transcriptional control of IFNα-stimulated genes, as well as more recent data on the regulation of transcription of genes stimulated by IFN-γ. The present disclosure is further illustrated by the identification of related genes encoding protein factors responsive to as yet unknown factors. It is expected that the murine 91 kD protein is responsive to IFN-γ.

For example, the above represents evidence that the 91 kD protein is the tyrosine kinase target when IFNγ is the ligand. Thus two different ligands acting through two different receptors both use these family members. With only a modest number of family members and combinatorial use in response to different ligands, this family of proteins becomes an even more likely possibility to represent a general link between ligand-occupied receptors and transcriptional control of specific genes in the nucleus.

It is proposed and shown by the foregoing that other members of the 113-91 protein family will be and have been identified as phosphorylation targets in response to other ligands. If as is believed, the tyrosine phosphorylation site on proteins in this family is conserved, one can then easily determine which family members are activated (phosphorylated), and likewise the particular extracellular polypeptide ligand to which that family member is responding. The modifications of these proteins (phosphorylation and dephosphorylation) enables the preparation and use of assays for determining the effectiveness of pharmaceuticals in potentiating or preventing intracellular responses to various polypeptides, and such assays are accordingly contemplated within the scope of the present invention.

Earlier work has concluded that DNA binding protein was activated in the cell cytoplasm in response to IFN-γ treatment and that this protein stimulated transcription of the GBP gene (10,14). In the present work, with the aid of antisera to proteins originally studied in connection with IFN-α gene stimulation (7,12,15), the 91 kD ISGF-3 protein has been assigned a prominent role in IFN-γ gene stimulation as well. The evidence for this conclusion included: 1) antisera specific to the 91 kD protein affected the IFN-γ dependent gel-shift complex, and 2) A 91 kD protein could be cross-linked to the GAS IFN-γ activated site. 3) A ³⁵ S-labeled 91 kD protein and a 91 kD immunoreactive protein specifically purified with the gel-shift complex. 4) The 91 kD protein is an IFN-γ dependent tyrosine kinase substrate as indeed it had earlier proved to be in response to IFN-α (15). 5) The 91 kD protein but not the 113 kD protein moved to the nucleus in response to IFN-γ treatment. None of these experiments prove but do strongly suggest that the same 91 kD protein acts differently in different DNA binding complexes that are triggered by either IFN-α or IFN-γ.

These results strongly support the hypothesis originated from studies on IFN-α that polypeptide cell surface receptors report their occupation by extracellular ligand to latent cytoplasmic proteins that after activation move to the nucleus to trigger transcription (4,15,21). Furthermore, because cytoplasmic phosphorylation and factor activation is so rapid it appears likely that the functional receptor complexes contain tyrosine kinase activity. Since the IFN-γ receptor chain that has been cloned thus far (22) has no hint of possessing intrinsic kinase activity, perhaps some other molecule with tyrosine kinase activity couples with the IFN-γ receptor. Two recent results with other receptors suggest possible parallels to the situation with the IFN receptors. The trk protein which has an intracellular tyrosine kinase domain, associates with the NGF receptor when that receptor is occupied (23). In addition, the lck protein, a member of the src family of tyrosine kinases, is co-precipitated with the T cell receptor (24). It is possible to predict that signal transduction to the nucleus through these two receptors could involve latent cytoplasmic substrates that form part of activated transcription factors. In any event, it seems possible that there are kinases like trk or lck associated with the IFN-γ receptor or with IFN-α receptor.

With regard to the effect of phosphorylation on the 91 kD protein, it was something of a surprise that after IFN-γ treatment the 91 kD protein becomes a DNA binding protein. Its role must be different in response to IFN-α treatment. Tyrosine is also phosphorylated on tyrosine and joins a complex with the 113 and 84 kD proteins but as judged by UV cross-linking studies (7), the 91 kD protein does not contact DNA.

In addition to becoming a DNA binding protein it is clear that the 91 kD protein is specifically translocated the nucleus in the wake of IFN-γ stimulation.

EXAMPLE 6

Dimerization of Phosphorylated STAT91

Stat91 (a 91 kD protein that acts as a signal transducer and activator of transcription) is inactive in the cytoplasm of untreated cells but is activated by phosphorylation on tyrosine in response to a number of polypeptide ligands including IFN-α and IFN-γ. This example reports that inactive Stat91 in the cytoplasm of untreated cells is a monomer and upon IFN-γ induced phosphorylation it forms a stable homodimer. The dimer is capable of binding to a specific DNA sequence directing transcription. Dissociation and reassociation assays show that dimerization of Stat91 is mediated through SH2-phosphotyrosyl peptide interactions. Dimerization involving SH2 recognition of specific phosphotyrosyl peptides may well provide a prototype for interactions among family members of STAT proteins to form different transcription complexes and Jak2 for the IFN-γ pathway (42, 43, 44). These kinases themselves become tyrosine phosphorylated to carry out specific signaling events.

MATERIALS AND METHODS

Cell Culture. Human 2fTGH, U3A cells were maintained in DMEM medium supplied with 10% bovine calf serum. U3A cell lines supplemented with various Stat91 protein constructs were maintained in 0.1 mg/ml G418 (Gibco, BRL).

Stable cell lines were selected as described (45). IFN-γ(5 ng/ml, gift from Amgen) treatment of cells was for 15 min. unless otherwise noted.

Plasmid Constructions. Expression construct MNC-84 was made by insertion of the cDNA into the Not I-Bam HI cloning site of an expression vector PMNC (45, 35). MNC-91L was made by insertion of the Stat91 cDNA into the Not I -Bam HI cloning sites of pMNC without the stop codon at the end, resulting the production of a long form of Stat91 with a C-terminal tag of 34 amino acids encoded by PMNC vector.

GST fusion protein expression plasmids were constructed by the using the pGEX-2T vector (Pharmacia). GST-91SH2 encodes amino acids 573 to 672 of Stat91; GST-91mSH2 encodes amino acids 573 to 672 of Stat91 with an Arg-602→Leu-602 mutation; and GST-91SH3 encodes amino acids 506 to 564 of Stat91.

DNA Transfection. DNA transfection was carried by the calcium phosphate method, and stable cell lines were selected in Dulbecco's modified Eagle's medium containing G418 (0.5 mg/ml, Gibco), as described (45).

Preparation of Cell Extracts. Crude whole cell extracts were prepared as described (31). Cytoplasmic and nuclear extracts were prepared essentially as described (46).

Affinity Purification. Affinity purification with a biotinylated oligonucleotide was described (31). The sequence of the biotinylated GAS oligonucleotide was from the Ly6E gene promoter (34).

Nondenaturing Potyacrylamide Gel Analysis. A nondenatured protein molecular weight marker kit with a range of molecular weights from 14 to 545 kD was obtained from Sigma. Determining molecular weights using nondenaturing polyacrylamide gel was carried out following the manufacturer's procedure, which is a modification of the methods of Bryan and Davis (47, 48). Phosphorylated and unphosphorylated Stat91 samples obtained from affinity purification using a biotinylated GAS oligonucleotide (31) were resuspended in a buffer containing 10 mM Tris (pH 6.7), 16% glycerol, 0.04% bromphenol blue (BPB). The mixtures were analyzed on 4.5%, 5.5%, 6.5%, and 7.5% native gels side by side with standard markers using a Bio-Rad mini-Protean II Cell electrophoresis system. Electrophoresis was stopped when the dye (BPB) reached the bottom of the gels. The molecular size markers were revealed by Coomassie blue staining. Phosphorylated and unphosphorylated Stat91 samples were detected by immunoblotting with anti-91T.

Glycerol Gradient Analysis. Cells extracts (Bud 8) were mixed with protein standards (Pharmacia) and subjected to centrifugation through preformed 10%-40% glycerol gradients for 40 hours at 40,000 rpm in an SW41 rotor as described (6).

Gel Mobility Shift Assays. Gel mobility shift assays were carried out as described (34). An oligonucleotide corresponding to the GAS element from the human FcγRI receptor gene (Pearse et al. 1993) was synthesized and used for gel mobility shift assays. The oligonucleotide has the following sequence: 5'GATCGAGATGTATTTCCCAGAAAAG3' (SEQ. ID NO:17).

Synthesis of Peptides. Solid phase peptide synthesis was used with either a DuPont RAMPS multiple synthesizer or by manual synthesis. C-terminal amino attached to Wang resin were obtained from DuPont/NEN. All amino acids were coupled as the N-Fmoc pentafluorophenyl esters (Advanced Chemtech), except for N-Fmoc, PO-dimethyl-L-phosphotyrosine (Bachem). Double couplings were used. Cleavage from resin and deprotection used thioanisol/m-creso/TFA/TMSBr at 4° C. for 16 hr. Purification used C-18 column HPLC with 0.1% TFA/acetonitrile gradients. Peptides were characterized by ¹ H and ³¹ P NMR, and by Mass Spec, and were greater than 95% pure.

Guanidium Hydrochloride Treatment. Extracts were incubated with guanidium hydrochloride (final concentration was 0.4 to 0.6 M) for two min. at room temperature and then diluted with gel shift buffer (final concentration of guanidium hydrochloride was 100 mM) and incubated at room temperature for 15 min. ³² P-labeled GAS oligonucleotide probe was then added directly to the mixture followed by gel mobility shift assay.

Dissociation-reassociation Analysis. Extracts were incubated with various concentrations of peptides or fusion proteins, and ³² P-labeled GAS oligonucleotide probe in gel shift buffer was then added to promote the formation of protein-DNA complex followed by mobility shift analysis. This assay did not involve guanidium hydrochloride treatment.

Preparation of Fusion Proteins. Bacterially expressed GST fusion proteins were purified using standard techniques, as described in Birge et al., 1992. Fusion proteins were quantified by O.D. absorbance at 280 nm. Aliquotes were frozen at -70° C.

RESULTS

Detection of Ligand Induced Dimer Fountain of Stat91 in Solution. In untreated cells, Stat91 is not phosphorylated on tyrosine. Treatment with IFN-γ leads within minutes to tyrosine phosphorylation and activation of DNA binding capacity. The phosphorylated form migrates more slowly during electrophoresis under denaturing conditions affording a simple assay for the phosphoprotein (31).

To determine the native molecular weights of the phosphorylated and unphosphorylated forms of Stat91, we separated them by affinity purification using a biotinylated deoxyoligonucleotide containing a GAS sequence (interferon gamma activation site) (FIG. 16A). The separation of phosphorylated Stat91 from the unphosphorylated form was efficient as almost all detectable phosphorylated form could bind to the GAS site while unphosphorylated Stat91 remained unbound. To determine the molecular weights of the purified phosphorylated Stat91 and unphosphorylated Stat91, samples of each were then subjected to electrophoresis through a set of nondenaturing gels containing various concentrations of acrylamide followed by Western blot analysis (FIG. 16B). Native protein size markers (Sigma) were included in the analysis.

This technique was originally described by Bryan (48) and was recently used for dimer analysis (49). The logic of the technique is that increasing gel concentrations affect the migration of larger proteins more than smaller proteins, and the analysis is not affected by modifications such as protein phosphorylation (49).

A function of the relative mobilities (Rm) was plotted versus the concentration of acrylamide for each sample to construct Ferguson plots (FIG. 16C). The logarithm of the retardation coefficient (calculated from FIG. 16C) of each sample was then plotted against the logarithm of the relevant molecular weight range (FIG. 16D). By extrapolation of its retardation coefficient (FIG. 16D), the native molecular weight of Stat91 from untreated cells was estimated to be approximately 95 kD, while tyrosine phosphorylated Stat91 was estimated to be about twice as large, or approximately 180 kD. Because the calculated molecular weight from amino acid sequence of Stat91 is 87 kD, and Stat91 migrates on denaturing SDA gels with an apparent molecular weight of 91 kD (see supra, and refs. 12 and 45), we concluded that in solution, unphosphorylated Stat91 existed as a monomer while tyrosine phosphorylated Stat91 is a dimer.

We also employed glycerol gradient analysis to estimate the native molecular weights of both phosphorylated and unphosphorylated Stat91 (FIG. 17). Whole cell extract of fibroblast cells (Bud8) treated with IFN-γ were prepared and subjected to sedimentation through a 10-40% glycerol gradient. Fractions from the gradient were collected and analyzed by both immunoblotting and gel mobility shift analysis (FIGS. 17A and 17B). As expected, two electrophoretic forms of Stat91 could be detected by immunoblotting (FIG. 17A): the slow-migrating form (tyrosine phosphorylated) and the fast-migrating form (unphosphorylated; FIG. 17A). The phosphorylated Stat91 sedimented more rapidly than the unphosphorylated form. Again, using molecular weight markers, the native molecular weight of the unphosphorylated form of Stat91 appeared to be about 90 kD while the tyrosine phosphorylated form of Stat91 was about 180 kD (FIG. 17C), supporting the conclusion that unphosphorylated Stat91 existed as a monomer in solution while the tyrosine phosphorylated form exists as a dimer. When fractions from the glycerol gradients were analyzed by electrophoretic mobility shift analysis (FIG. 17B), the peak of the phosphorylated form of Stat91correlated well with the DNA-binding activity of Stat91. Thus only the phosphorylated dimeric Stat91 has the sequence-specific DNA recognition capacity.

Stat91 Binds DNA as a Dimer. Long or short versions of DNA binding protein can produce, respectively, a slower or a faster migrating band during gel retardation assays. Finding intermediate gel shift bands produced by mixing two different sized species provides evidence of dimerization of the DNA binding proteins. Since Stat91 requires specific tyrosine phosphorylation in ligand-treated cells for its DNA binding, we sought evidence of formation of such heterodimers, first in transfected cells. An expression vector (MNC911) encoding Stat91L, a recombinant form of Stat91 containing an additional 34 amino acid carboxyl terminal tag was generated. [The extra amino acids were encoded by a segment of DNA sequence from plasmid pMNC (see Materials and Methods).] A Stat84 expression vector (MNC84) was also available (45). From somatic cell genetic experiments, mutant human cell lines (U3) are known that lack the Stat91/84 mRNA and proteins (29,30). The U3 cells were therefore separately transfected with vectors encoding Stat84 (MNC84) or Stat91L (MNC91L) or a mixture of both vectors. Permanent transfectants expressing Stat84 (C84), Stat91L (C91L) or both proteins (Cmx) were isolated (FIG. 18A).

Mobility shift analysis was performed with extracts from these stable cell lines (FIG. 18B). Extracts of IFN-γ-treated C84 cells produced a faster migrating gel shift band than extracts of treated C91L cells. Most importantly, extracts from IFN-γ-treated Cmx cells expressing both Stat84 and Stat91L proteins formed an additional intermediate gel shift band. Anti-91, an antiserum against the C-terminal 38 amino acids of Stat91 (12) that are absent in Stat84, specifically removed the top two shift bands seen with the Cmx extracts. Anti-91, an antiserum against amino acids 609 to 716 (15) that recognizes both Stat91L and Stat84, proteins inhibited the binding of all three shift bands. Thus, the middle band formed by extracts of the Cmx cells is clearly identified as a heterodimer of Stat84 and Stat91L. We concluded that both Stat91 and Stat84 bind DNA as homodimers and, if present in the same cell, will form heterodimers.

We next wanted to detect the formation of dimers in vitro. When cytoplasmic or nuclear extracts of IFN-γ-treated C84 or C91L cells were mixed and analyzed (FIG. 19), only the fast or slow migrating gel shift bands were observed. Thus it appeared that once formed in vivo, the dimers were stable. To promote the formation of protein interchange between the subunits of the dimer, a mixture of either cytoplasmic or nuclear extracts of IFN-γ-treated C84 or C91L cells were subjected mild denaturation-renaturation treatment: extracts were made 0.5 M with respect to guanidium hydrochloride for two minutes and then diluted for renaturation and subsequently used for gel retardation analysis. The formation of heterodimer was clearly detected after this treatment. When extracts from either C84 cells alone or C91L cells alone were subjected to the same treatment, the intermediate band did not form. The intermediate band was again proven by antiserum treatment to consist of Stat84/Stat91L dimer (data not shown).

This experiment defined conditions under which the dimer was stable, but also showed that dissociation and reassociation of the dimer in vitro was possible. Since guanidium hydrochloride is known to disrupt only non-covalent chemical bonds, it seemed that Stat91 (or Stat84) homodimerization was mediated through non-covalent interactions.

Dimenization of Stat91 Involves Phosphotyrosyl Peptide and SH2 Interactions. Based on the results described above, we devised a dissociation-reassociation assay in the absence of guanidium hydrochloride to explore the possible nature of interactions involved in dimer formation (FIG. 20). When the short and the long forms of a homodimer are mixed with a dissociating agent (e.g., a peptide containing the putative dimerization domain), the subunits of the dimer should dissociate (in a concentration dependent fashion) due to the interaction of the agent with the dimerization domain(s) of the protein. When a specific DNA probe is subsequently added to the mixture to drive the formation of a stable protein-DNA complex, the detection of any reassociated or remaining dimers can be assayed. In the presence of low concentration of the dissociating agent, addition of DNA to form the stable protein-DNA complex should lead to the detection of homodimers as well as heterodimers. At high concentration of the dissociating agent, subunits of the dimer may not be able to re-form and no DNA-protein complexes would be detected (FIG. 20).

The Stat91 sequence contains an SH2 domain (amino acids 569 to 700, see discussion below), and we knew that Tyr-701 was the single phosphorylated tyrosine residue required for DNA binding activity (supra, 45). Furthermore, we have observed that phosphotyrosine at 10 mM, but not phosphoserine or phosphothreonine, could prevent the formation of Stat91-DNA complex. We therefore sought evidence that the dimerization of Stat91 involved specific SH2-phosphotyrosine interaction using the dissociation and reassociation assay.

In order to evaluate the role of the SH2-phosphotyrosine interation, two peptides fragments of Stat91 corresponding to segments of the SH2 and phosphotyrosing domains of Stat91 were prepared: a non-phosphorylated peptide (91Y), LDGPKGTGYIKTELI (SEQ. ID NO:18) (corresponding to amino acids 693-707), and a phosphotyrosyl peptide (91Y-p), GY*IKTE (SEQ. ID NO:19) (representing residues 700-705).

Activated Stat84 or Stat91L was obtained from IFN-γ-treated C84 or C91L cells and mixed in the presence of various concentrations of the peptides followed by gel mobility shift analysis. The non-phosphorylated peptide had no effect on the presence of the two gel shift bands characteristic of Stat84 or Stat91L homodimers (FIG. 21, lane 2-4). In contrast, the phosphorylated peptide (91Y-p) at the concentration of 4 μM clearly promoted the exchange between the subunits of Stat84 dimers and Stat91L dimers to form heterodimers (FIG. 21, lane 5). At a higher concentration (160 μM), peptide 91Y-p but not the unphosphorylated peptide dissociated the dimers and blocked the formation of DNA protein complexes (FIG. 21, lane 7).

When cells are treated with IFN-α both Stat91 (or 84) and Stat113 become phosphorylated (15). Antiserum to Stat113 can precipitate both Stat113 and Stat91 after IFN-α-treatment but not before, suggesting IFN-α dependent interaction of these two proteins, perhaps as a heterodimer (15).

In Stat113, tyr-690 in the homologous position to Tyr-701 in Stat91 is the single target residue for phosphorylation. Amino acids downstream of the affected tyrosine residue show some homology between the two proteins. We therefore prepared a phosphotyrosyl peptide of Stat113 (113Y-p), KVNLQERRKY*LKHR (SEQ. ID NO:20) [amino acids 681 to 694; (38)]. At concentrations similar to 91Y-p, 113Y-p also promoted the exchange of subunits between the Stat84 and Stat91L, while at a high concentration (40 μM), 113Y-p prevented the gel shift bands almost completely (FIG. 21, lane 8-10).

We prepared a phosphotyrosyl peptide (SrcY-p), EPQY*EEIPIYL (SEQ. ID NO:21) which is known to interact with the Src SH2 domain with a high affinity (50). This peptide showed no effect on the Stat91 dimer formation (FIG. 21, lane 11-13). Thus, it seems that Stat91 dimerization involves SH2 interaction with tyrosine residues in specific peptide sequence.

To test further the specificity of Stat91 dimerization mediated through specific-phosphotyrosyl-peptide SH2 interaction, a fusion product of glutathione-S-transferase with the Stat91-SH2 domain (GST-91SH2) was prepared (FIG. 22A) and used in the in vitro dissociation reassociation assay. At concentrations of 0.5 to 5 μM, the Stat91-SH2 domain promoted the formation of a heterodimer (FIG. 22B, lanes 5-7). In contrast, neither GST alone, nor fusion products with a mutant (R⁶⁰² →L⁶⁰²) Stat91-SH2 domain (GST 91mSH2) that renders Stat91 non-functional in vivo, a Stat91 SH3 domain (GST-91SH3), nor the Src SH2 domain (GST-SrcSH2), induced the exchange of subunits between the Stat84 and Stat91L homodimers (FIG. 22B).

DISCUSSION

The initial sequence analysis of the Stat91 and Stat113 proteins revealed the presence of SH2 like domains (see 13,38). Further it was found that STAT proteins themselves are phosphorylated on single tyrosine residues during their activation (15,31). Single amino acid mutations either removing the Stat91 phosphorylation site, Tyr-701, or converting Arg-702 to Leu in the highly conserved "pocket" region of the SH2 domain abolished the activity of Stat91 (45). Thus it seemed highly likely that one possible role of the STAT SH2 domains would be to bind the phosphotyrosine residues in one of the JAK kinases.

Since the activated STATs hav e phosphotyrosine residues and SH2 domains, a second suggested role for SH2 domains was in protein--protein interactions within the STAT family. By two physical criteria--electrophoresis in native gels and sedimentation on gradients--Stat91 in untreated cells is a monomer and in treated cells is a dimer (FIGS. 16-18). Since phosphotyrosyl peptides from Stat91 or Stat113 and the SH2 domain of Stat91 could efficiently promote the formation of herterodimers between Stat91L and Stat84 in a disassociation and reassociation assay, we conclude that dimerization of Stat91 involves SH2-phosphotyrosyl peptide interactions.

The possibility of an SH2 domain in Stat91 was indicated initially by the presence of highly conserved amino acid stretches between the Stat91 and Stat113 sequences in the 569 to 700 residue region, several of which, especially the FLLR sequence in the amino terminal end of the region, are characteristic of -SH2 domains. The C-terminal half of the SH2 domains are less well conserved in general (39); this was also true for the STAT proteins compared to other proteins, although Stat91and Stat113 are quite similar in this region (38, 13, FIG. 23). The available structures of lck, src, abl, and p85a SH2's permit identification of structurally conserved regions (SCR's), and detailed alignment of amino acid sequences of several proteins (FIG. 23) is based on these.

The characteristic W (in βA1) is preceded by hydrophilic residues and is followed by hydrophobic residues in Stat91, but alignment to the W seems justified, even if the small beta sheet of which the W is part is shifted in Stat91. The three positively charged residues contributing to the phosphotyrosyl binding site are at the positions indicated as alphaA2, betaB5, and betaD5. FIG. 23 shows an alignment which accomplishes this by insertions in the `AA` and `CD` regions. This is a different alignment from that previously suggested (38), and gives a satisfactory alignment in the (beta)D region, although, like the previous alignment, it is obviously considerably less similar to the other SH2's in the C-terminus.

This alignment suggests that the SH2 domain in the Stat91 would end in the vicinity of residue 700. In such an alignment, the Tyr-701 occurs almost immediately after the SH2 domain: a distance too short to allow an intramolecular phosphotyrosine -SH2 interaction. Since the data presented earlier strongly implicate that an SH2-phosphotyrosine interaction is involved in dimerization, such an interaction is likely to be between two phospho Stat91 subunits as a reciprocal pTyr -SH2 interaction.

The apparent stability of Stat91 dimer may be due to a high association rate coupled with a high dissociation rate of SH2-phosphotyrosyl peptide interactions as suggested (Felder et al., 1993, Mol. Cell Biol. 13:1449-1455) coupled with interactions between other domains of Stat91 that may contribute stability to the Stat91 dimer. Interference by homologous phosphopeptides with the -SH2-phosphotyrosine interaction would then lower stability sufficiently to allow complete dissociation and heterodimerization.

The dimer formation between phospho Stat91 is the first case in eukaryotes where dimer formation is regulated by phosphorylation, and the only one thus far dependent on tyrosine phosphorylation. We anticipate that dimerization with the STAT protein family will be important. It seems likely that in cells treated with IFN-α, there is Stat113-Stat91 interaction (15). This may well be mediated through SH2 and phosphotyrosyl peptide interactions as described above, leading to a complex (a probable dimer of Stat91-Stat113) which joins with a 48 kD DNA binding protein (a member of another family of DNA binding factors) to make a complex capable of binding to a different DNA site. Furthermore, we have recently cloned two mouse cDNAs which encode other STAT family members that have conserved the same general structure features observed in the Stat91 and Stat113 molecules (see Example 5, Supra). (U.S. application Ser. No. 08/126,588, filed Sep. 29, 1993, which is specifically incorporated herein by reference in its entirety). Thus the specificity of STAT-containing complexes will almost surely be affected by which proteins are phosphorylated and then available for dimer formation.

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48. Bryan, J. K. (1977). Anal. Biochem. 78:513.

49. Amster-Choder, O. and Wright, A. (1992). Science 257:1395.

50. Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, et al. (1993). Cell 72:767.

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                 - (1) GENERAL INFORMATION:                                                     -    (iii) NUMBER OF SEQUENCES: 25                                             - (2) INFORMATION FOR SEQ ID NO:1:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 3268 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: unknown                                                -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Homo sapi - #ens                                       -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: HeLa                                                      -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION: 25..2577                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                  - ACTGCAACCC TAATCAGAGC CCAA ATG GCG CAG TGG GAA A - #TG CTG CAG AAT             51                                                                           #Met Leu Gln Asna Gln Trp Glu                                                  #        5  1                                                                  - CTT GAC AGC CCC TTT CAG GAT CAG CTG CAC CA - #G CTT TAC TCG CAC AGC            99                                                                           Leu Asp Ser Pro Phe Gln Asp Gln Leu His Gl - #n Leu Tyr Ser His Ser            # 25                                                                           - CTC CTG CCT GTG GAC ATT CGA CAG TAC TTG GC - #T GTC TGG ATT GAA GAC           147                                                                           Leu Leu Pro Val Asp Ile Arg Gln Tyr Leu Al - #a Val Trp Ile Glu Asp            #                 40                                                           - CAG AAC TGG CAG GAA GCT GCA CTT GGG AGT GA - #T GAT TCC AAG GCT ACC           195                                                                           Gln Asn Trp Gln Glu Ala Ala Leu Gly Ser As - #p Asp Ser Lys Ala Thr            #             55                                                               - ATG CTA TTC TTC CAC TTC TTG GAT CAG CTG AA - #C TAT GAG TGT GGC CGT           243                                                                           Met Leu Phe Phe His Phe Leu Asp Gln Leu As - #n Tyr Glu Cys Gly Arg            #         70                                                                   - TGC AGC CAG GAC CCA GAG TCC TTG TTG CTG CA - #G CAC AAT TTG CGG AAA           291                                                                           Cys Ser Gln Asp Pro Glu Ser Leu Leu Leu Gl - #n His Asn Leu Arg Lys            #     85                                                                       - TTC TGC CGG GAC ATT CAG CCC TTT TCC CAG GA - #T CCT ACC CAG TTG GCT           339                                                                           Phe Cys Arg Asp Ile Gln Pro Phe Ser Gln As - #p Pro Thr Gln Leu Ala            #105                                                                           - GAG ATG ATC TTT AAC CTC CTT CTG GAA GAA AA - #A AGA ATT TTG ATC CAG           387                                                                           Glu Met Ile Phe Asn Leu Leu Leu Glu Glu Ly - #s Arg Ile Leu Ile Gln            #               120                                                            - GCT CAG AGG GCC CAA TTG GAA CAA GGA GAG CC - #A GTT CTC GAA ACA CCT           435                                                                           Ala Gln Arg Ala Gln Leu Glu Gln Gly Glu Pr - #o Val Leu Glu Thr Pro            #           135                                                                - GTG GAG AGC CAG CAA CAT GAG ATT GAA TCC CG - #G ATC CTG GAT TTA AGG           483                                                                           Val Glu Ser Gln Gln His Glu Ile Glu Ser Ar - #g Ile Leu Asp Leu Arg            #       150                                                                    - GCT ATG ATG GAG AAG CTG GTA AAA TCC ATC AG - #C CAA CTG AAA GAC CAG           531                                                                           Ala Met Met Glu Lys Leu Val Lys Ser Ile Se - #r Gln Leu Lys Asp Gln            #   165                                                                        - CAG GAT GTC TTC TGC TTC CGA TAT AAG ATC CA - #G GCC AAA GGG AAG ACA           579                                                                           Gln Asp Val Phe Cys Phe Arg Tyr Lys Ile Gl - #n Ala Lys Gly Lys Thr            170                 1 - #75                 1 - #80                 1 -        #85                                                                            - CCC TCT CTG GAC CCC CAT CAG ACC AAA GAG CA - #G AAG ATT CTG CAG GAA           627                                                                           Pro Ser Leu Asp Pro His Gln Thr Lys Glu Gl - #n Lys Ile Leu Gln Glu            #               200                                                            - ACT CTC AAT GAA CTG GAC AAA AGG AGA AAG GA - #G GTG CTG GAT GCC TCC           675                                                                           Thr Leu Asn Glu Leu Asp Lys Arg Arg Lys Gl - #u Val Leu Asp Ala Ser            #           215                                                                - AAA GCA CTG CTA GGC CGA TTA ACT ACC CTA AT - #C GAG CTA CTG CTG CCA           723                                                                           Lys Ala Leu Leu Gly Arg Leu Thr Thr Leu Il - #e Glu Leu Leu Leu Pro            #       230                                                                    - AAG TTG GAG GAG TGG AAG GCC CAG CAG CAA AA - #A GCC TGC ATC AGA GCT           771                                                                           Lys Leu Glu Glu Trp Lys Ala Gln Gln Gln Ly - #s Ala Cys Ile Arg Ala            #   245                                                                        - CCC ATT GAC CAC GGG TTG GAA CAG CTG GAG AC - #A TGG TTC ACA GCT GGA           819                                                                           Pro Ile Asp His Gly Leu Glu Gln Leu Glu Th - #r Trp Phe Thr Ala Gly            250                 2 - #55                 2 - #60                 2 -        #65                                                                            - GCA AAG CTG TTG TTT CAC CTG AGG CAG CTG CT - #G AAG GAG CTG AAG GGA           867                                                                           Ala Lys Leu Leu Phe His Leu Arg Gln Leu Le - #u Lys Glu Leu Lys Gly            #               280                                                            - CTG AGT TGC CTG GTT AGC TAT CAG GAT GAC CC - #T CTG ACC AAA GGG GTG           915                                                                           Leu Ser Cys Leu Val Ser Tyr Gln Asp Asp Pr - #o Leu Thr Lys Gly Val            #           295                                                                - GAC CTA CGC AAC GCC CAG GTC ACA GAG TTG CT - #A CAG CGT CTG CTC CAC           963                                                                           Asp Leu Arg Asn Ala Gln Val Thr Glu Leu Le - #u Gln Arg Leu Leu His            #       310                                                                    - AGA GCC TTT GTG GTA GAA ACC CAG CCC TGC AT - #G CCC CAA ACT CCC CAT          1011                                                                           Arg Ala Phe Val Val Glu Thr Gln Pro Cys Me - #t Pro Gln Thr Pro His            #   325                                                                        - CGA CCC CTC ATC CTC AAG ACT GGC AGC AAG TT - #C ACC GTC CGA ACA AGG          1059                                                                           Arg Pro Leu Ile Leu Lys Thr Gly Ser Lys Ph - #e Thr Val Arg Thr Arg            330                 3 - #35                 3 - #40                 3 -        #45                                                                            - CTG CTG GTG AGA CTC CAG GAA GGC AAT GAG TC - #A CTG ACT GTG GAA GTC          1107                                                                           Leu Leu Val Arg Leu Gln Glu Gly Asn Glu Se - #r Leu Thr Val Glu Val            #               360                                                            - TCC ATT GAC AGG AAT CCT CCT CAA TTA CAA GG - #C TTC CGG AAG TTC AAC          1155                                                                           Ser Ile Asp Arg Asn Pro Pro Gln Leu Gln Gl - #y Phe Arg Lys Phe Asn            #           375                                                                - ATT CTG ACT TCA AAC CAG AAA ACT TTG ACC CC - #C GAG AAG GGG CAG AGT          1203                                                                           Ile Leu Thr Ser Asn Gln Lys Thr Leu Thr Pr - #o Glu Lys Gly Gln Ser            #       390                                                                    - CAG GGT TTG ATT TGG GAC TTT GGT TAC CTG AC - #T CTG GTG GAG CAA CGT          1251                                                                           Gln Gly Leu Ile Trp Asp Phe Gly Tyr Leu Th - #r Leu Val Glu Gln Arg            #   405                                                                        - TCA GGT GGT TCA GGA AAG GGC AGC AAT AAG GG - #G CCA CTA GGT GTG ACA          1299                                                                           Ser Gly Gly Ser Gly Lys Gly Ser Asn Lys Gl - #y Pro Leu Gly Val Thr            410                 4 - #15                 4 - #20                 4 -        #25                                                                            - GAG GAA CTG CAC ATC ATC AGC TTC ACG GTC AA - #A TAT ACC TAC CAG GGT          1347                                                                           Glu Glu Leu His Ile Ile Ser Phe Thr Val Ly - #s Tyr Thr Tyr Gln Gly            #               440                                                            - CTG AAG CAG GAG CTG AAA ACG GAC ACC CTC CC - #T GTG GTG ATT ATT TCC          1395                                                                           Leu Lys Gln Glu Leu Lys Thr Asp Thr Leu Pr - #o Val Val Ile Ile Ser            #           455                                                                - AAC ATG AAC CAG CTC TCA ATT GCC TGG GCT TC - #A GTT CTC TGG TTC AAT          1443                                                                           Asn Met Asn Gln Leu Ser Ile Ala Trp Ala Se - #r Val Leu Trp Phe Asn            #       470                                                                    - TTG CTC AGC CCA AAC CTT CAG AAC CAG CAG TT - #C TTC TCC AAC CCC CCC          1491                                                                           Leu Leu Ser Pro Asn Leu Gln Asn Gln Gln Ph - #e Phe Ser Asn Pro Pro            #   485                                                                        - AAG GCC CCC TGG AGC TTG CTG GGC CCT GCT CT - #C AGT TGG CAG TTC TCC          1539                                                                           Lys Ala Pro Trp Ser Leu Leu Gly Pro Ala Le - #u Ser Trp Gln Phe Ser            490                 4 - #95                 5 - #00                 5 -        #05                                                                            - TCC TAT GTT GGC CGA GGC CTC AAC TCA GAC CA - #G CTG AGC ATG CTG AGA          1587                                                                           Ser Tyr Val Gly Arg Gly Leu Asn Ser Asp Gl - #n Leu Ser Met Leu Arg            #               520                                                            - AAC AAG CTG TTC GGG CAG AAC TGT AGG ACT GA - #G GAT CCA TTA TTG TCC          1635                                                                           Asn Lys Leu Phe Gly Gln Asn Cys Arg Thr Gl - #u Asp Pro Leu Leu Ser            #           535                                                                - TGG GCT GAC TTC ACT AAG CGA GAG AGC CCT CC - #T GGC AAG TTA CCA TTC          1683                                                                           Trp Ala Asp Phe Thr Lys Arg Glu Ser Pro Pr - #o Gly Lys Leu Pro Phe            #       550                                                                    - TGG ACA TGG CTG GAC AAA ATT CTG GAG TTG GT - #A CAT GAC CAC CTG AAG          1731                                                                           Trp Thr Trp Leu Asp Lys Ile Leu Glu Leu Va - #l His Asp His Leu Lys            #   565                                                                        - GAT CTC TGG AAT GAT GGA CGC ATC ATG GGC TT - #T GTG AGT CGG AGC CAG          1779                                                                           Asp Leu Trp Asn Asp Gly Arg Ile Met Gly Ph - #e Val Ser Arg Ser Gln            570                 5 - #75                 5 - #80                 5 -        #85                                                                            - GAG CGC CGG CTG CTG AAG AAG ACC ATG TCT GG - #C ACC TTT CTA CTG CGC          1827                                                                           Glu Arg Arg Leu Leu Lys Lys Thr Met Ser Gl - #y Thr Phe Leu Leu Arg            #               600                                                            - TTC AGT GAA TCG TCA GAA GGG GGC ATT ACC TG - #C TCC TGG GTG GAG CAC          1875                                                                           Phe Ser Glu Ser Ser Glu Gly Gly Ile Thr Cy - #s Ser Trp Val Glu His            #           615                                                                - CAG GAT GAT GAC AAG GTG CTC ATC TAC TCT GT - #G CAA CCG TAC ACG AAG          1923                                                                           Gln Asp Asp Asp Lys Val Leu Ile Tyr Ser Va - #l Gln Pro Tyr Thr Lys            #       630                                                                    - GAG GTG CTG CAG TCA CTC CCG CTG ACT GAA AT - #C ATC CGC CAT TAC CAG          1971                                                                           Glu Val Leu Gln Ser Leu Pro Leu Thr Glu Il - #e Ile Arg His Tyr Gln            #   645                                                                        - TTG CTC ACT GAG GAG AAT ATA CCT GAA AAC CC - #A CTG CGC TTC CTC TAT          2019                                                                           Leu Leu Thr Glu Glu Asn Ile Pro Glu Asn Pr - #o Leu Arg Phe Leu Tyr            650                 6 - #55                 6 - #60                 6 -        #65                                                                            - CCC CGA ATC CCC CGG GAT GAA GCT TTT GGG TG - #C TAC TAC CAG GAG AAA          2067                                                                           Pro Arg Ile Pro Arg Asp Glu Ala Phe Gly Cy - #s Tyr Tyr Gln Glu Lys            #               680                                                            - GTT AAT CTC CAG GAA CGG AGG AAA TAC CTG AA - #A CAC AGG CTC ATT GTG          2115                                                                           Val Asn Leu Gln Glu Arg Arg Lys Tyr Leu Ly - #s His Arg Leu Ile Val            #           695                                                                - GTC TCT AAT AGA CAG GTG GAT GAA CTG CAA CA - #A CCG CTG GAG CTT AAG          2163                                                                           Val Ser Asn Arg Gln Val Asp Glu Leu Gln Gl - #n Pro Leu Glu Leu Lys            #       710                                                                    - CCA GAG CCA GAG CTG GAG TCA TTA GAG CTG GA - #A CTA GGG CTG GTG CCA          2211                                                                           Pro Glu Pro Glu Leu Glu Ser Leu Glu Leu Gl - #u Leu Gly Leu Val Pro            #   725                                                                        - GAG CCA GAG CTC AGC CTG GAC TTA GAG CCA CT - #G CTG AAG GCA GGG CTG          2259                                                                           Glu Pro Glu Leu Ser Leu Asp Leu Glu Pro Le - #u Leu Lys Ala Gly Leu            730                 7 - #35                 7 - #40                 7 -        #45                                                                            - GAT CTG GGG CCA GAG CTA GAG TCT GTG CTG GA - #G TCC ACT CTG GAG CCT          2307                                                                           Asp Leu Gly Pro Glu Leu Glu Ser Val Leu Gl - #u Ser Thr Leu Glu Pro            #               760                                                            - GTG ATA GAG CCC ACA CTA TGC ATG GTA TCA CA - #A ACA GTG CCA GAG CCA          2355                                                                           Val Ile Glu Pro Thr Leu Cys Met Val Ser Gl - #n Thr Val Pro Glu Pro            #           775                                                                - GAC CAA GGA CCT GTA TCA CAG CCA GTG CCA GA - #G CCA GAT TTG CCC TGT          2403                                                                           Asp Gln Gly Pro Val Ser Gln Pro Val Pro Gl - #u Pro Asp Leu Pro Cys            #       790                                                                    - GAT CTG AGA CAT TTG AAC ACT GAG CCA ATG GA - #A ATC TTC AGA AAC TGT          2451                                                                           Asp Leu Arg His Leu Asn Thr Glu Pro Met Gl - #u Ile Phe Arg Asn Cys            #   805                                                                        - GTA AAG ATT GAA GAA ATC ATG CCG AAT GGT GA - #C CCA CTG TTG GCT GGC          2499                                                                           Val Lys Ile Glu Glu Ile Met Pro Asn Gly As - #p Pro Leu Leu Ala Gly            810                 8 - #15                 8 - #20                 8 -        #25                                                                            - CAG AAC ACC GTG GAT GAG GTT TAC GTC TCC CG - #C CCC AGC CAC TTC TAC          2547                                                                           Gln Asn Thr Val Asp Glu Val Tyr Val Ser Ar - #g Pro Ser His Phe Tyr            #               840                                                            - ACT GAT GGA CCC TTG ATG CCT TCT GAC TTC TA - #GGAACCAC ATTTCCTCTG            2597                                                                           Thr Asp Gly Pro Leu Met Pro Ser Asp Phe                                        #           850                                                                - TTCTTTTCAT ATCTCTTTGC CCTTCCTACT CCTCATAGCA TGATATTGTT CT - #CCAAGGAT        2657                                                                           - GGGAATCAGG CATGTGTCCC TTCCAAGCTG TGTTAACTGT TCAAACTCAG GC - #CTGTGTGA        2717                                                                           - CTCCATTGGG GTGAGAGGTG AAAGCATAAC ATGGGTACAG AGGGGACAAC AA - #TGAATCAG        2777                                                                           - AACAGATGCT GAGCCATAGG TCTAAATAGG ATCCTGGAGG CTGCCTGCTG TG - #CTGGGAGG        2837                                                                           - TATAGGGGTC CTGGGGGCAG GCCAGGGCAG TTGACAGGTA CTTGGAGGGC TC - #AGGGCAGT        2897                                                                           - GGCTTCTTTC CAGTATGGAA GGATTTCAAC ATTTTAATAG TTGGTTAGGC TA - #AACTGGTG        2957                                                                           - CATACTGGCA TTGGCCTTGG TGGGGAGCAC AGACACAGGA TAGGACTCCA TT - #TCTTTCTT        3017                                                                           - CCATTCCTTC ATGTCTAGGA TAACTTGCTT TCTTCTTTCC TTTACTCCTG GC - #TCAAGCCC        3077                                                                           - TGAATTTCTT CTTTTCCTGC AGGGGTTGAG AGCTTTCTGC CTTAGCCTAC CA - #TGTGAAAC        3137                                                                           - TCTACCCTGA AGAAAGGGAT GGATAGGAAG TAGACCTCTT TTTCTTACCA GT - #CTCCTCCC        3197                                                                           - CTACTCTGCC CCCTAAGCTG GCTGTACCTG TTCCTCCCCC ATAAAATGAT CC - #TGCCAATC        3257                                                                           #     3268                                                                     - (2) INFORMATION FOR SEQ ID NO:2:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 851 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                  - Met Ala Gln Trp Glu Met Leu Gln Asn Leu As - #p Ser Pro Phe Gln Asp          #                 15                                                           - Gln Leu His Gln Leu Tyr Ser His Ser Leu Le - #u Pro Val Asp Ile Arg          #             30                                                               - Gln Tyr Leu Ala Val Trp Ile Glu Asp Gln As - #n Trp Gln Glu Ala Ala          #         45                                                                   - Leu Gly Ser Asp Asp Ser Lys Ala Thr Met Le - #u Phe Phe His Phe Leu          #     60                                                                       - Asp Gln Leu Asn Tyr Glu Cys Gly Arg Cys Se - #r Gln Asp Pro Glu Ser          # 80                                                                           - Leu Leu Leu Gln His Asn Leu Arg Lys Phe Cy - #s Arg Asp Ile Gln Pro          #                 95                                                           - Phe Ser Gln Asp Pro Thr Gln Leu Ala Glu Me - #t Ile Phe Asn Leu Leu          #           110                                                                - Leu Glu Glu Lys Arg Ile Leu Ile Gln Ala Gl - #n Arg Ala Gln Leu Glu          #       125                                                                    - Gln Gly Glu Pro Val Leu Glu Thr Pro Val Gl - #u Ser Gln Gln His Glu          #   140                                                                        - Ile Glu Ser Arg Ile Leu Asp Leu Arg Ala Me - #t Met Glu Lys Leu Val          145                 1 - #50                 1 - #55                 1 -        #60                                                                            - Lys Ser Ile Ser Gln Leu Lys Asp Gln Gln As - #p Val Phe Cys Phe Arg          #               175                                                            - Tyr Lys Ile Gln Ala Lys Gly Lys Thr Pro Se - #r Leu Asp Pro His Gln          #           190                                                                - Thr Lys Glu Gln Lys Ile Leu Gln Glu Thr Le - #u Asn Glu Leu Asp Lys          #       205                                                                    - Arg Arg Lys Glu Val Leu Asp Ala Ser Lys Al - #a Leu Leu Gly Arg Leu          #   220                                                                        - Thr Thr Leu Ile Glu Leu Leu Leu Pro Lys Le - #u Glu Glu Trp Lys Ala          225                 2 - #30                 2 - #35                 2 -        #40                                                                            - Gln Gln Gln Lys Ala Cys Ile Arg Ala Pro Il - #e Asp His Gly Leu Glu          #               255                                                            - Gln Leu Glu Thr Trp Phe Thr Ala Gly Ala Ly - #s Leu Leu Phe His Leu          #           270                                                                - Arg Gln Leu Leu Lys Glu Leu Lys Gly Leu Se - #r Cys Leu Val Ser Tyr          #       285                                                                    - Gln Asp Asp Pro Leu Thr Lys Gly Val Asp Le - #u Arg Asn Ala Gln Val          #   300                                                                        - Thr Glu Leu Leu Gln Arg Leu Leu His Arg Al - #a Phe Val Val Glu Thr          305                 3 - #10                 3 - #15                 3 -        #20                                                                            - Gln Pro Cys Met Pro Gln Thr Pro His Arg Pr - #o Leu Ile Leu Lys Thr          #               335                                                            - Gly Ser Lys Phe Thr Val Arg Thr Arg Leu Le - #u Val Arg Leu Gln Glu          #           350                                                                - Gly Asn Glu Ser Leu Thr Val Glu Val Ser Il - #e Asp Arg Asn Pro Pro          #       365                                                                    - Gln Leu Gln Gly Phe Arg Lys Phe Asn Ile Le - #u Thr Ser Asn Gln Lys          #   380                                                                        - Thr Leu Thr Pro Glu Lys Gly Gln Ser Gln Gl - #y Leu Ile Trp Asp Phe          385                 3 - #90                 3 - #95                 4 -        #00                                                                            - Gly Tyr Leu Thr Leu Val Glu Gln Arg Ser Gl - #y Gly Ser Gly Lys Gly          #               415                                                            - Ser Asn Lys Gly Pro Leu Gly Val Thr Glu Gl - #u Leu His Ile Ile Ser          #           430                                                                - Phe Thr Val Lys Tyr Thr Tyr Gln Gly Leu Ly - #s Gln Glu Leu Lys Thr          #       445                                                                    - Asp Thr Leu Pro Val Val Ile Ile Ser Asn Me - #t Asn Gln Leu Ser Ile          #   460                                                                        - Ala Trp Ala Ser Val Leu Trp Phe Asn Leu Le - #u Ser Pro Asn Leu Gln          465                 4 - #70                 4 - #75                 4 -        #80                                                                            - Asn Gln Gln Phe Phe Ser Asn Pro Pro Lys Al - #a Pro Trp Ser Leu Leu          #               495                                                            - Gly Pro Ala Leu Ser Trp Gln Phe Ser Ser Ty - #r Val Gly Arg Gly Leu          #           510                                                                - Asn Ser Asp Gln Leu Ser Met Leu Arg Asn Ly - #s Leu Phe Gly Gln Asn          #       525                                                                    - Cys Arg Thr Glu Asp Pro Leu Leu Ser Trp Al - #a Asp Phe Thr Lys Arg          #   540                                                                        - Glu Ser Pro Pro Gly Lys Leu Pro Phe Trp Th - #r Trp Leu Asp Lys Ile          545                 5 - #50                 5 - #55                 5 -        #60                                                                            - Leu Glu Leu Val His Asp His Leu Lys Asp Le - #u Trp Asn Asp Gly Arg          #               575                                                            - Ile Met Gly Phe Val Ser Arg Ser Gln Glu Ar - #g Arg Leu Leu Lys Lys          #           590                                                                - Thr Met Ser Gly Thr Phe Leu Leu Arg Phe Se - #r Glu Ser Ser Glu Gly          #       605                                                                    - Gly Ile Thr Cys Ser Trp Val Glu His Gln As - #p Asp Asp Lys Val Leu          #   620                                                                        - Ile Tyr Ser Val Gln Pro Tyr Thr Lys Glu Va - #l Leu Gln Ser Leu Pro          625                 6 - #30                 6 - #35                 6 -        #40                                                                            - Leu Thr Glu Ile Ile Arg His Tyr Gln Leu Le - #u Thr Glu Glu Asn Ile          #               655                                                            - Pro Glu Asn Pro Leu Arg Phe Leu Tyr Pro Ar - #g Ile Pro Arg Asp Glu          #           670                                                                - Ala Phe Gly Cys Tyr Tyr Gln Glu Lys Val As - #n Leu Gln Glu Arg Arg          #       685                                                                    - Lys Tyr Leu Lys His Arg Leu Ile Val Val Se - #r Asn Arg Gln Val Asp          #   700                                                                        - Glu Leu Gln Gln Pro Leu Glu Leu Lys Pro Gl - #u Pro Glu Leu Glu Ser          705                 7 - #10                 7 - #15                 7 -        #20                                                                            - Leu Glu Leu Glu Leu Gly Leu Val Pro Glu Pr - #o Glu Leu Ser Leu Asp          #               735                                                            - Leu Glu Pro Leu Leu Lys Ala Gly Leu Asp Le - #u Gly Pro Glu Leu Glu          #           750                                                                - Ser Val Leu Glu Ser Thr Leu Glu Pro Val Il - #e Glu Pro Thr Leu Cys          #       765                                                                    - Met Val Ser Gln Thr Val Pro Glu Pro Asp Gl - #n Gly Pro Val Ser Gln          #   780                                                                        - Pro Val Pro Glu Pro Asp Leu Pro Cys Asp Le - #u Arg His Leu Asn Thr          785                 7 - #90                 7 - #95                 8 -        #00                                                                            - Glu Pro Met Glu Ile Phe Arg Asn Cys Val Ly - #s Ile Glu Glu Ile Met          #               815                                                            - Pro Asn Gly Asp Pro Leu Leu Ala Gly Gln As - #n Thr Val Asp Glu Val          #           830                                                                - Tyr Val Ser Arg Pro Ser His Phe Tyr Thr As - #p Gly Pro Leu Met Pro          #       845                                                                    - Ser Asp Phe                                                                      850                                                                        - (2) INFORMATION FOR SEQ ID NO:3:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 3943 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: unknown                                                -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Homo sapi - #ens                                       -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: Human Stat91                                              -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION: 197..2449                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                  - ATTAAACCTC TCGCCGAGCC CCTCCGCAGA CTCTGCGCCG GAAAGTTTCA TT - #TGCTGTAT          60                                                                           - GCCATCCTCG AGAGCTGTCT AGGTTAACGT TCGCACTCTG TGTATATAAC CT - #CGACAGTC         120                                                                           - TTGGCACCTA ACGTGCTGTG CGTAGCTGCT CCTTTGGTTG AATCCCCAGG CC - #CTTGTTGG         180                                                                           #CAG CAG CTT GAC        229 CAG TGG TAC GAA CTT                                #Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu A - #sp                               #                 10                                                           - TCA AAA TTC CTG GAG CAG GTT CAC CAG CTT TA - #T GAT GAC AGT TTT CCC           277                                                                           Ser Lys Phe Leu Glu Gln Val His Gln Leu Ty - #r Asp Asp Ser Phe Pro            #             25                                                               - ATG GAA ATC AGA CAG TAC CTG GCA CAG TGG TT - #A GAA AAG CAA GAC TGG           325                                                                           Met Glu Ile Arg Gln Tyr Leu Ala Gln Trp Le - #u Glu Lys Gln Asp Trp            #         40                                                                   - GAG CAC GCT GCC AAT GAT GTT TCA TTT GCC AC - #C ATC CGT TTT CAT GAC           373                                                                           Glu His Ala Ala Asn Asp Val Ser Phe Ala Th - #r Ile Arg Phe His Asp            #     55                                                                       - CTC CTG TCA CAG CTG GAT GAT CAA TAT AGT CG - #C TTT TCT TTG GAG AAT           421                                                                           Leu Leu Ser Gln Leu Asp Asp Gln Tyr Ser Ar - #g Phe Ser Leu Glu Asn            # 75                                                                           - AAC TTC TTG CTA CAG CAT AAC ATA AGG AAA AG - #C AAG CGT AAT CTT CAG           469                                                                           Asn Phe Leu Leu Gln His Asn Ile Arg Lys Se - #r Lys Arg Asn Leu Gln            #                 90                                                           - GAT AAT TTT CAG GAA GAC CCA ATC CAG ATG TC - #T ATG ATC ATT TAC AGC           517                                                                           Asp Asn Phe Gln Glu Asp Pro Ile Gln Met Se - #r Met Ile Ile Tyr Ser            #            105                                                               - TGT CTG AAG GAA GAA AGG AAA ATT CTG GAA AA - #C GCC CAG AGA TTT AAT           565                                                                           Cys Leu Lys Glu Glu Arg Lys Ile Leu Glu As - #n Ala Gln Arg Phe Asn            #       120                                                                    - CAG GCT CAG TCG GGG AAT ATT CAG AGC ACA GT - #G ATG TTA GAC AAA CAG           613                                                                           Gln Ala Gln Ser Gly Asn Ile Gln Ser Thr Va - #l Met Leu Asp Lys Gln            #   135                                                                        - AAA GAG CTT GAC AGT AAA GTC AGA AAT GTG AA - #G GAC AAG GTT ATG TGT           661                                                                           Lys Glu Leu Asp Ser Lys Val Arg Asn Val Ly - #s Asp Lys Val Met Cys            140                 1 - #45                 1 - #50                 1 -        #55                                                                            - ATA GAG CAT GAA ATC AAG AGC CTG GAA GAT TT - #A CAA GAT GAA TAT GAC           709                                                                           Ile Glu His Glu Ile Lys Ser Leu Glu Asp Le - #u Gln Asp Glu Tyr Asp            #               170                                                            - TTC AAA TGC AAA ACC TTG CAG AAC AGA GAA CA - #C GAG ACC AAT GGT GTG           757                                                                           Phe Lys Cys Lys Thr Leu Gln Asn Arg Glu Hi - #s Glu Thr Asn Gly Val            #           185                                                                - GCA AAG AGT GAT CAG AAA CAA GAA CAG CTG TT - #A CTC AAG AAG ATG TAT           805                                                                           Ala Lys Ser Asp Gln Lys Gln Glu Gln Leu Le - #u Leu Lys Lys Met Tyr            #       200                                                                    - TTA ATG CTT GAC AAT AAG AGA AAG GAA GTA GT - #T CAC AAA ATA ATA GAG           853                                                                           Leu Met Leu Asp Asn Lys Arg Lys Glu Val Va - #l His Lys Ile Ile Glu            #   215                                                                        - TTG CTG AAT GTC ACT GAA CTT ACC CAG AAT GC - #C CTG ATT AAT GAT GAA           901                                                                           Leu Leu Asn Val Thr Glu Leu Thr Gln Asn Al - #a Leu Ile Asn Asp Glu            220                 2 - #25                 2 - #30                 2 -        #35                                                                            - CTA GTG GAG TGG AAG CGG AGA CAG CAG AGC GC - #C TGT ATT GGG GGG CCG           949                                                                           Leu Val Glu Trp Lys Arg Arg Gln Gln Ser Al - #a Cys Ile Gly Gly Pro            #               250                                                            - CCC AAT GCT TGC TTG GAT CAG CTG CAG AAC TG - #G TTC ACT ATA GTT GCG           997                                                                           Pro Asn Ala Cys Leu Asp Gln Leu Gln Asn Tr - #p Phe Thr Ile Val Ala            #           265                                                                - GAG AGT CTG CAG CAA GTT CGG CAG CAG CTT AA - #A AAG TTG GAG GAA TTG          1045                                                                           Glu Ser Leu Gln Gln Val Arg Gln Gln Leu Ly - #s Lys Leu Glu Glu Leu            #       280                                                                    - GAA CAG AAA TAC ACC TAC GAA CAT GAC CCT AT - #C ACA AAA AAC AAA CAA          1093                                                                           Glu Gln Lys Tyr Thr Tyr Glu His Asp Pro Il - #e Thr Lys Asn Lys Gln            #   295                                                                        - GTG TTA TGG GAC CGC ACC TTC AGT CTT TTC CA - #G CAG CTC ATT CAG AGC          1141                                                                           Val Leu Trp Asp Arg Thr Phe Ser Leu Phe Gl - #n Gln Leu Ile Gln Ser            300                 3 - #05                 3 - #10                 3 -        #15                                                                            - TCG TTT GTG GTG GAA AGA CAG CCC TGC ATG CC - #A ACG CAC CCT CAG AGG          1189                                                                           Ser Phe Val Val Glu Arg Gln Pro Cys Met Pr - #o Thr His Pro Gln Arg            #               330                                                            - CCG CTG GTC TTG AAG ACA GGG GTC CAG TTC AC - #T GTG AAG TTG AGA CTG          1237                                                                           Pro Leu Val Leu Lys Thr Gly Val Gln Phe Th - #r Val Lys Leu Arg Leu            #           345                                                                - TTG GTG AAA TTG CAA GAG CTG AAT TAT AAT TT - #G AAA GTC AAA GTC TTA          1285                                                                           Leu Val Lys Leu Gln Glu Leu Asn Tyr Asn Le - #u Lys Val Lys Val Leu            #       360                                                                    - TTT GAT AAA GAT GTG AAT GAG AGA AAT ACA GT - #A AAA GGA TTT AGG AAG          1333                                                                           Phe Asp Lys Asp Val Asn Glu Arg Asn Thr Va - #l Lys Gly Phe Arg Lys            #   375                                                                        - TTC AAC ATT TTG GGC ACG CAC ACA AAA GTG AT - #G AAC ATG GAG GAG TCC          1381                                                                           Phe Asn Ile Leu Gly Thr His Thr Lys Val Me - #t Asn Met Glu Glu Ser            380                 3 - #85                 3 - #90                 3 -        #95                                                                            - ACC AAT GGC AGT CTG GCG GCT GAA TTT CGG CA - #C CTG CAA TTG AAA GAA          1429                                                                           Thr Asn Gly Ser Leu Ala Ala Glu Phe Arg Hi - #s Leu Gln Leu Lys Glu            #               410                                                            - CAG AAA AAT GCT GGC ACC AGA ACG AAT GAG GG - #T CCT CTC ATC GTT ACT          1477                                                                           Gln Lys Asn Ala Gly Thr Arg Thr Asn Glu Gl - #y Pro Leu Ile Val Thr            #           425                                                                - GAA GAG CTT CAC TCC CTT AGT TTT GAA ACC CA - #A TTG TGC CAG CCT GGT          1525                                                                           Glu Glu Leu His Ser Leu Ser Phe Glu Thr Gl - #n Leu Cys Gln Pro Gly            #       440                                                                    - TTG GTA ATT GAC CTC GAG ACG ACC TCT CTG CC - #C GTT GTG GTG ATC TCC          1573                                                                           Leu Val Ile Asp Leu Glu Thr Thr Ser Leu Pr - #o Val Val Val Ile Ser            #   455                                                                        - AAC GTC AGC CAG CTC CCG AGC GGT TGG GCC TC - #C ATC CTT TGG TAC AAC          1621                                                                           Asn Val Ser Gln Leu Pro Ser Gly Trp Ala Se - #r Ile Leu Trp Tyr Asn            460                 4 - #65                 4 - #70                 4 -        #75                                                                            - ATG CTG GTG GCG GAA CCC AGG AAT CTG TCC TT - #C TTC CTG ACT CCA CCA          1669                                                                           Met Leu Val Ala Glu Pro Arg Asn Leu Ser Ph - #e Phe Leu Thr Pro Pro            #               490                                                            - TGT GCA CGA TGG GCT CAG CTT TCA GAA GTG CT - #G AGT TGG CAG TTT TCT          1717                                                                           Cys Ala Arg Trp Ala Gln Leu Ser Glu Val Le - #u Ser Trp Gln Phe Ser            #           505                                                                - TCT GTC ACC AAA AGA GGT CTC AAT GTG GAC CA - #G CTG AAC ATG TTG GGA          1765                                                                           Ser Val Thr Lys Arg Gly Leu Asn Val Asp Gl - #n Leu Asn Met Leu Gly            #       520                                                                    - GAG AAG CTT CTT GGT CCT AAC GCC AGC CCC GA - #T GGT CTC ATT CCG TGG          1813                                                                           Glu Lys Leu Leu Gly Pro Asn Ala Ser Pro As - #p Gly Leu Ile Pro Trp            #   535                                                                        - ACG AGG TTT TGT AAG GAA AAT ATA AAT GAT AA - #A AAT TTT CCC TTC TGG          1861                                                                           Thr Arg Phe Cys Lys Glu Asn Ile Asn Asp Ly - #s Asn Phe Pro Phe Trp            540                 5 - #45                 5 - #50                 5 -        #55                                                                            - CTT TGG ATT GAA AGC ATC CTA GAA CTC ATT AA - #A AAA CAC CTG CTC CCT          1909                                                                           Leu Trp Ile Glu Ser Ile Leu Glu Leu Ile Ly - #s Lys His Leu Leu Pro            #               570                                                            - CTC TGG AAT GAT GGG TGC ATC ATG GGC TTC AT - #C AGC AAG GAG CGA GAG          1957                                                                           Leu Trp Asn Asp Gly Cys Ile Met Gly Phe Il - #e Ser Lys Glu Arg Glu            #           585                                                                - CGT GCC CTG TTG AAG GAC CAG CAG CCG GGG AC - #C TTC CTG CTG CGG TTC          2005                                                                           Arg Ala Leu Leu Lys Asp Gln Gln Pro Gly Th - #r Phe Leu Leu Arg Phe            #       600                                                                    - AGT GAG AGC TCC CGG GAA GGG GCC ATC ACA TT - #C ACA TGG GTG GAG CGG          2053                                                                           Ser Glu Ser Ser Arg Glu Gly Ala Ile Thr Ph - #e Thr Trp Val Glu Arg            #   615                                                                        - TCC CAG AAC GGA GGC GAA CCT GAC TTC CAT GC - #G GTT GAA CCC TAC ACG          2101                                                                           Ser Gln Asn Gly Gly Glu Pro Asp Phe His Al - #a Val Glu Pro Tyr Thr            620                 6 - #25                 6 - #30                 6 -        #35                                                                            - AAG AAA GAA CTT TCT GCT GTT ACT TTC CCT GA - #C ATC ATT CGC AAT TAC          2149                                                                           Lys Lys Glu Leu Ser Ala Val Thr Phe Pro As - #p Ile Ile Arg Asn Tyr            #               650                                                            - AAA GTC ATG GCT GCT GAG AAT ATT CCT GAG AA - #T CCC CTG AAG TAT CTG          2197                                                                           Lys Val Met Ala Ala Glu Asn Ile Pro Glu As - #n Pro Leu Lys Tyr Leu            #           665                                                                - TAT CCA AAT ATT GAC AAA GAC CAT GCC TTT GG - #A AAG TAT TAC TCC AGG          2245                                                                           Tyr Pro Asn Ile Asp Lys Asp His Ala Phe Gl - #y Lys Tyr Tyr Ser Arg            #       680                                                                    - CCA AAG GAA GCA CCA GAG CCA ATG GAA CTT GA - #T GGC CCT AAA GGA ACT          2293                                                                           Pro Lys Glu Ala Pro Glu Pro Met Glu Leu As - #p Gly Pro Lys Gly Thr            #   695                                                                        - GGA TAT ATC AAG ACT GAG TTG ATT TCT GTG TC - #T GAA GTT CAC CCT TCT          2341                                                                           Gly Tyr Ile Lys Thr Glu Leu Ile Ser Val Se - #r Glu Val His Pro Ser            700                 7 - #05                 7 - #10                 7 -        #15                                                                            - AGA CTT CAG ACC ACA GAC AAC CTG CTC CCC AT - #G TCT CCT GAG GAG TTT          2389                                                                           Arg Leu Gln Thr Thr Asp Asn Leu Leu Pro Me - #t Ser Pro Glu Glu Phe            #               730                                                            - GAC GAG GTG TCT CGG ATA GTG GGC TCT GTA GA - #A TTC GAC AGT ATG ATG          2437                                                                           Asp Glu Val Ser Arg Ile Val Gly Ser Val Gl - #u Phe Asp Ser Met Met            #           745                                                                - AAC ACA GTA TAGAGCATGA ATTTTTTTCA TCTTCTCTGG CGACAGTTT - #T                  2486                                                                           Asn Thr Val                                                                            750                                                                    - CCTTCTCATC TGTGATTCCC TCCTGCTACT CTGTTCCTTC ACATCCTGTG TT - #TCTAGGGA        2546                                                                           - AATGAAAGAA AGGCCAGCAA ATTCGCTGCA ACCTGTTGAT AGCAAGTGAA TT - #TTTCTCTA        2606                                                                           - ACTCAGAAAC ATCAGTTACT CTGAAGGGCA TCATGCATCT TACTGAAGGT AA - #AATTGAAA        2666                                                                           - GGCATTCTCT GAAGAGTGGG TTTCACAAGT GAAAAACATC CAGATACACC CA - #AAGTATCA        2726                                                                           - GGACGAGAAT GAGGGTCCTT TGGGAAAGGA GAAGTTAAGC AACATCTAGC AA - #ATGTTATG        2786                                                                           - CATAAAGTCA GTGCCCAACT GTTATAGGTT GTTGGATAAA TCAGTGGTTA TT - #TAGGGAAC        2846                                                                           - TGCTTGACGT AGGAACGGTA AATTTCTGTG GGAGAATTCT TACATGTTTT CT - #TTGCTTTA        2906                                                                           - AGTGTAACTG GCAGTTTTCC ATTGGTTTAC CTGTGAAATA GTTCAAAGCC AA - #GTTTATAT        2966                                                                           - ACAATTATAT CAGTCCTCTT TCAAAGGTAG CCATCATGGA TCTGGTAGGG GG - #AAAATGTG        3026                                                                           - TATTTTATTA CATCTTTCAC ATTGGCTATT TAAAGACAAA GACAAATTCT GT - #TTCTTGAG        3086                                                                           - AAGAGAACAT TTCCAAATTC ACAAGTTGTG TTTGATATCC AAAGCTGAAT AC - #ATTCTGCT        3146                                                                           - TTCATCTTGG TCACATACAA TTATTTTTAC AGTTCTCCCA AGGGAGTTAG GC - #TATTCACA        3206                                                                           - ACCACTCATT CAAAAGTTGA AATTAACCAT AGATGTAGAT AAACTCAGAA AT - #TTAATTCA        3266                                                                           - TGTTTCTTAA ATGGGCTACT TTGTCCTTTT TGTTATTAGG GTGGTATTTA GT - #CTATTAGC        3326                                                                           - CACAAAATTG GGAAAGGAGT AGAAAAAGCA GTAACTGACA ACTTGAATAA TA - #CACCAGAG        3386                                                                           - ATAATATGAG AATCAGATCA TTTCAAAACT CATTTCCTAT GTAACTGCAT TG - #AGAACTGC        3446                                                                           - ATATGTTTCG CTGATATATG TGTTTTTCAC ATTTGCGAAT GGTTCCATTC TC - #TCTCCTGT        3506                                                                           - ACTTTTTCCA GACACTTTTT TGAGTGGATG ATGTTTCGTG AAGTATACTG TA - #TTTTTACC        3566                                                                           - TTTTTCCTTC CTTATCACTG ACACAAAAAG TAGATTAAGA GATGGGTTTG AC - #AAGGTTCT        3626                                                                           - TCCCTTTTAC ATACTGCTGT CTATGTGGCT GTATCTTGTT TTTCCACTAC TG - #CTACCACA        3686                                                                           - ACTATATTAT CATGCAAATG CTGTATTCTT CTTTGGTGGA GATAAAGATT TC - #TTGAGTTT        3746                                                                           - TGTTTTAAAA TTAAAGCTAA AGTATCTGTA TTGCATTAAA TATAATATCG AC - #ACAGTGCT        3806                                                                           - TTCCGTGGCA CTGCATACAA TCTGAGGCCT CCTCTCTCAG TTTTTATATA GA - #TGGCGAGA        3866                                                                           - ACCTAAGTTT CAGTTGATTT TACAATTGAA ATGACTAAAA AACAAAGAAG AC - #AACATTAA        3926                                                                           # 3943             A                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 750 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                  - Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu As - #p Ser Lys Phe Leu Glu          #                 15                                                           - Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pr - #o Met Glu Ile Arg Gln          #             30                                                               - Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Tr - #p Glu His Ala Ala Asn          #         45                                                                   - Asp Val Ser Phe Ala Thr Ile Arg Phe His As - #p Leu Leu Ser Gln Leu          #     60                                                                       - Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu As - #n Asn Phe Leu Leu Gln          # 80                                                                           - His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gl - #n Asp Asn Phe Gln Glu          #                 95                                                           - Asp Pro Ile Gln Met Ser Met Ile Ile Tyr Se - #r Cys Leu Lys Glu Glu          #           110                                                                - Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe As - #n Gln Ala Gln Ser Gly          #       125                                                                    - Asn Ile Gln Ser Thr Val Met Leu Asp Lys Gl - #n Lys Glu Leu Asp Ser          #   140                                                                        - Lys Val Arg Asn Val Lys Asp Lys Val Met Cy - #s Ile Glu His Glu Ile          145                 1 - #50                 1 - #55                 1 -        #60                                                                            - Lys Ser Leu Glu Asp Leu Gln Asp Glu Tyr As - #p Phe Lys Cys Lys Thr          #               175                                                            - Leu Gln Asn Arg Glu His Glu Thr Asn Gly Va - #l Ala Lys Ser Asp Gln          #           190                                                                - Lys Gln Glu Gln Leu Leu Leu Lys Lys Met Ty - #r Leu Met Leu Asp Asn          #       205                                                                    - Lys Arg Lys Glu Val Val His Lys Ile Ile Gl - #u Leu Leu Asn Val Thr          #   220                                                                        - Glu Leu Thr Gln Asn Ala Leu Ile Asn Asp Gl - #u Leu Val Glu Trp Lys          225                 2 - #30                 2 - #35                 2 -        #40                                                                            - Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pr - #o Pro Asn Ala Cys Leu          #               255                                                            - Asp Gln Leu Gln Asn Trp Phe Thr Ile Val Al - #a Glu Ser Leu Gln Gln          #           270                                                                - Val Arg Gln Gln Leu Lys Lys Leu Glu Glu Le - #u Glu Gln Lys Tyr Thr          #       285                                                                    - Tyr Glu His Asp Pro Ile Thr Lys Asn Lys Gl - #n Val Leu Trp Asp Arg          #   300                                                                        - Thr Phe Ser Leu Phe Gln Gln Leu Ile Gln Se - #r Ser Phe Val Val Glu          305                 3 - #10                 3 - #15                 3 -        #20                                                                            - Arg Gln Pro Cys Met Pro Thr His Pro Gln Ar - #g Pro Leu Val Leu Lys          #               335                                                            - Thr Gly Val Gln Phe Thr Val Lys Leu Arg Le - #u Leu Val Lys Leu Gln          #           350                                                                - Glu Leu Asn Tyr Asn Leu Lys Val Lys Val Le - #u Phe Asp Lys Asp Val          #       365                                                                    - Asn Glu Arg Asn Thr Val Lys Gly Phe Arg Ly - #s Phe Asn Ile Leu Gly          #   380                                                                        - Thr His Thr Lys Val Met Asn Met Glu Glu Se - #r Thr Asn Gly Ser Leu          385                 3 - #90                 3 - #95                 4 -        #00                                                                            - Ala Ala Glu Phe Arg His Leu Gln Leu Lys Gl - #u Gln Lys Asn Ala Gly          #               415                                                            - Thr Arg Thr Asn Glu Gly Pro Leu Ile Val Th - #r Glu Glu Leu His Ser          #           430                                                                - Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gl - #y Leu Val Ile Asp Leu          #       445                                                                    - Glu Thr Thr Ser Leu Pro Val Val Val Ile Se - #r Asn Val Ser Gln Leu          #   460                                                                        - Pro Ser Gly Trp Ala Ser Ile Leu Trp Tyr As - #n Met Leu Val Ala Glu          465                 4 - #70                 4 - #75                 4 -        #80                                                                            - Pro Arg Asn Leu Ser Phe Phe Leu Thr Pro Pr - #o Cys Ala Arg Trp Ala          #               495                                                            - Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Se - #r Ser Val Thr Lys Arg          #           510                                                                - Gly Leu Asn Val Asp Gln Leu Asn Met Leu Gl - #y Glu Lys Leu Leu Gly          #       525                                                                    - Pro Asn Ala Ser Pro Asp Gly Leu Ile Pro Tr - #p Thr Arg Phe Cys Lys          #   540                                                                        - Glu Asn Ile Asn Asp Lys Asn Phe Pro Phe Tr - #p Leu Trp Ile Glu Ser          545                 5 - #50                 5 - #55                 5 -        #60                                                                            - Ile Leu Glu Leu Ile Lys Lys His Leu Leu Pr - #o Leu Trp Asn Asp Gly          #               575                                                            - Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Gl - #u Arg Ala Leu Leu Lys          #           590                                                                - Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Ph - #e Ser Glu Ser Ser Arg          #       605                                                                    - Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Ar - #g Ser Gln Asn Gly Gly          #   620                                                                        - Glu Pro Asp Phe His Ala Val Glu Pro Tyr Th - #r Lys Lys Glu Leu Ser          625                 6 - #30                 6 - #35                 6 -        #40                                                                            - Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Ty - #r Lys Val Met Ala Ala          #               655                                                            - Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Le - #u Tyr Pro Asn Ile Asp          #           670                                                                - Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Ar - #g Pro Lys Glu Ala Pro          #       685                                                                    - Glu Pro Met Glu Leu Asp Gly Pro Lys Gly Th - #r Gly Tyr Ile Lys Thr          #   700                                                                        - Glu Leu Ile Ser Val Ser Glu Val His Pro Se - #r Arg Leu Gln Thr Thr          705                 7 - #10                 7 - #15                 7 -        #20                                                                            - Asp Asn Leu Leu Pro Met Ser Pro Glu Glu Ph - #e Asp Glu Val Ser Arg          #               735                                                            - Ile Val Gly Ser Val Glu Phe Asp Ser Met Me - #t Asn Thr Val                  #           750                                                                - (2) INFORMATION FOR SEQ ID NO:5:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 2607 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: unknown                                                -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Homo sapi - #ens                                       -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION: 197..2335                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                  - ATTAAACCTC TCGCCGAGCC CCTCCGCAGA CTCTGCGCCG GAAAGTTTCA TT - #TGCTGTAT          60                                                                           - GCCATCCTCG AGAGCTGTCT AGGTTAACGT TCGCACTCTG TGTATATAAC CT - #CGACAGTC         120                                                                           - TTGGCACCTA ACGTGCTGTG CGTAGCTGCT CCTTTGGTTG AATCCCCAGG CC - #CTTGTTGG         180                                                                           #CAG CAG CTT GAC        229 CAG TGG TAC GAA CTT                                #Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu A - #sp                               #                 10                                                           - TCA AAA TTC CTG GAG CAG GTT CAC CAG CTT TA - #T GAT GAC AGT TTT CCC           277                                                                           Ser Lys Phe Leu Glu Gln Val His Gln Leu Ty - #r Asp Asp Ser Phe Pro            #             25                                                               - ATG GAA ATC AGA CAG TAC CTG GCA CAG TGG TT - #A GAA AAG CAA GAC TGG           325                                                                           Met Glu Ile Arg Gln Tyr Leu Ala Gln Trp Le - #u Glu Lys Gln Asp Trp            #         40                                                                   - GAG CAC GCT GCC AAT GAT GTT TCA TTT GCC AC - #C ATC CGT TTT CAT GAC           373                                                                           Glu His Ala Ala Asn Asp Val Ser Phe Ala Th - #r Ile Arg Phe His Asp            #     55                                                                       - CTC CTG TCA CAG CTG GAT GAT CAA TAT AGT CG - #C TTT TCT TTG GAG AAT           421                                                                           Leu Leu Ser Gln Leu Asp Asp Gln Tyr Ser Ar - #g Phe Ser Leu Glu Asn            # 75                                                                           - AAC TTC TTG CTA CAG CAT AAC ATA AGG AAA AG - #C AAG CGT AAT CTT CAG           469                                                                           Asn Phe Leu Leu Gln His Asn Ile Arg Lys Se - #r Lys Arg Asn Leu Gln            #                 90                                                           - GAT AAT TTT CAG GAA GAC CCA ATC CAG ATG TC - #T ATG ATC ATT TAC AGC           517                                                                           Asp Asn Phe Gln Glu Asp Pro Ile Gln Met Se - #r Met Ile Ile Tyr Ser            #            105                                                               - TGT CTG AAG GAA GAA AGG AAA ATT CTG GAA AA - #C GCC CAG AGA TTT AAT           565                                                                           Cys Leu Lys Glu Glu Arg Lys Ile Leu Glu As - #n Ala Gln Arg Phe Asn            #       120                                                                    - CAG GCT CAG TCG GGG AAT ATT CAG AGC ACA GT - #G ATG TTA GAC AAA CAG           613                                                                           Gln Ala Gln Ser Gly Asn Ile Gln Ser Thr Va - #l Met Leu Asp Lys Gln            #   135                                                                        - AAA GAG CTT GAC AGT AAA GTC AGA AAT GTG AA - #G GAC AAG GTT ATG TGT           661                                                                           Lys Glu Leu Asp Ser Lys Val Arg Asn Val Ly - #s Asp Lys Val Met Cys            140                 1 - #45                 1 - #50                 1 -        #55                                                                            - ATA GAG CAT GAA ATC AAG AGC CTG GAA GAT TT - #A CAA GAT GAA TAT GAC           709                                                                           Ile Glu His Glu Ile Lys Ser Leu Glu Asp Le - #u Gln Asp Glu Tyr Asp            #               170                                                            - TTC AAA TGC AAA ACC TTG CAG AAC AGA GAA CA - #C GAG ACC AAT GGT GTG           757                                                                           Phe Lys Cys Lys Thr Leu Gln Asn Arg Glu Hi - #s Glu Thr Asn Gly Val            #           185                                                                - GCA AAG AGT GAT CAG AAA CAA GAA CAG CTG TT - #A CTC AAG AAG ATG TAT           805                                                                           Ala Lys Ser Asp Gln Lys Gln Glu Gln Leu Le - #u Leu Lys Lys Met Tyr            #       200                                                                    - TTA ATG CTT GAC AAT AAG AGA AAG GAA GTA GT - #T CAC AAA ATA ATA GAG           853                                                                           Leu Met Leu Asp Asn Lys Arg Lys Glu Val Va - #l His Lys Ile Ile Glu            #   215                                                                        - TTG CTG AAT GTC ACT GAA CTT ACC CAG AAT GC - #C CTG ATT AAT GAT GAA           901                                                                           Leu Leu Asn Val Thr Glu Leu Thr Gln Asn Al - #a Leu Ile Asn Asp Glu            220                 2 - #25                 2 - #30                 2 -        #35                                                                            - CTA GTG GAG TGG AAG CGG AGA CAG CAG AGC GC - #C TGT ATT GGG GGG CCG           949                                                                           Leu Val Glu Trp Lys Arg Arg Gln Gln Ser Al - #a Cys Ile Gly Gly Pro            #               250                                                            - CCC AAT GCT TGC TTG GAT CAG CTG CAG AAC TG - #G TTC ACT ATA GTT GCG           997                                                                           Pro Asn Ala Cys Leu Asp Gln Leu Gln Asn Tr - #p Phe Thr Ile Val Ala            #           265                                                                - GAG AGT CTG CAG CAA GTT CGG CAG CAG CTT AA - #A AAG TTG GAG GAA TTG          1045                                                                           Glu Ser Leu Gln Gln Val Arg Gln Gln Leu Ly - #s Lys Leu Glu Glu Leu            #       280                                                                    - GAA CAG AAA TAC ACC TAC GAA CAT GAC CCT AT - #C ACA AAA AAC AAA CAA          1093                                                                           Glu Gln Lys Tyr Thr Tyr Glu His Asp Pro Il - #e Thr Lys Asn Lys Gln            #   295                                                                        - GTG TTA TGG GAC CGC ACC TTC AGT CTT TTC CA - #G CAG CTC ATT CAG AGC          1141                                                                           Val Leu Trp Asp Arg Thr Phe Ser Leu Phe Gl - #n Gln Leu Ile Gln Ser            300                 3 - #05                 3 - #10                 3 -        #15                                                                            - TCG TTT GTG GTG GAA AGA CAG CCC TGC ATG CC - #A ACG CAC CCT CAG AGG          1189                                                                           Ser Phe Val Val Glu Arg Gln Pro Cys Met Pr - #o Thr His Pro Gln Arg            #               330                                                            - CCG CTG GTC TTG AAG ACA GGG GTC CAG TTC AC - #T GTG AAG TTG AGA CTG          1237                                                                           Pro Leu Val Leu Lys Thr Gly Val Gln Phe Th - #r Val Lys Leu Arg Leu            #           345                                                                - TTG GTG AAA TTG CAA GAG CTG AAT TAT AAT TT - #G AAA GTC AAA GTC TTA          1285                                                                           Leu Val Lys Leu Gln Glu Leu Asn Tyr Asn Le - #u Lys Val Lys Val Leu            #       360                                                                    - TTT GAT AAA GAT GTG AAT GAG AGA AAT ACA GT - #A AAA GGA TTT AGG AAG          1333                                                                           Phe Asp Lys Asp Val Asn Glu Arg Asn Thr Va - #l Lys Gly Phe Arg Lys            #   375                                                                        - TTC AAC ATT TTG GGC ACG CAC ACA AAA GTG AT - #G AAC ATG GAG GAG TCC          1381                                                                           Phe Asn Ile Leu Gly Thr His Thr Lys Val Me - #t Asn Met Glu Glu Ser            380                 3 - #85                 3 - #90                 3 -        #95                                                                            - ACC AAT GGC AGT CTG GCG GCT GAA TTT CGG CA - #C CTG CAA TTG AAA GAA          1429                                                                           Thr Asn Gly Ser Leu Ala Ala Glu Phe Arg Hi - #s Leu Gln Leu Lys Glu            #               410                                                            - CAG AAA AAT GCT GGC ACC AGA ACG AAT GAG GG - #T CCT CTC ATC GTT ACT          1477                                                                           Gln Lys Asn Ala Gly Thr Arg Thr Asn Glu Gl - #y Pro Leu Ile Val Thr            #           425                                                                - GAA GAG CTT CAC TCC CTT AGT TTT GAA ACC CA - #A TTG TGC CAG CCT GGT          1525                                                                           Glu Glu Leu His Ser Leu Ser Phe Glu Thr Gl - #n Leu Cys Gln Pro Gly            #       440                                                                    - TTG GTA ATT GAC CTC GAG ACG ACC TCT CTG CC - #C GTT GTG GTG ATC TCC          1573                                                                           Leu Val Ile Asp Leu Glu Thr Thr Ser Leu Pr - #o Val Val Val Ile Ser            #   455                                                                        - AAC GTC AGC CAG CTC CCG AGC GGT TGG GCC TC - #C ATC CTT TGG TAC AAC          1621                                                                           Asn Val Ser Gln Leu Pro Ser Gly Trp Ala Se - #r Ile Leu Trp Tyr Asn            460                 4 - #65                 4 - #70                 4 -        #75                                                                            - ATG CTG GTG GCG GAA CCC AGG AAT CTG TCC TT - #C TTC CTG ACT CCA CCA          1669                                                                           Met Leu Val Ala Glu Pro Arg Asn Leu Ser Ph - #e Phe Leu Thr Pro Pro            #               490                                                            - TGT GCA CGA TGG GCT CAG CTT TCA GAA GTG CT - #G AGT TGG CAG TTT TCT          1717                                                                           Cys Ala Arg Trp Ala Gln Leu Ser Glu Val Le - #u Ser Trp Gln Phe Ser            #           505                                                                - TCT GTC ACC AAA AGA GGT CTC AAT GTG GAC CA - #G CTG AAC ATG TTG GGA          1765                                                                           Ser Val Thr Lys Arg Gly Leu Asn Val Asp Gl - #n Leu Asn Met Leu Gly            #       520                                                                    - GAG AAG CTT CTT GGT CCT AAC GCC AGC CCC GA - #T GGT CTC ATT CCG TGG          1813                                                                           Glu Lys Leu Leu Gly Pro Asn Ala Ser Pro As - #p Gly Leu Ile Pro Trp            #   535                                                                        - ACG AGG TTT TGT AAG GAA AAT ATA AAT GAT AA - #A AAT TTT CCC TTC TGG          1861                                                                           Thr Arg Phe Cys Lys Glu Asn Ile Asn Asp Ly - #s Asn Phe Pro Phe Trp            540                 5 - #45                 5 - #50                 5 -        #55                                                                            - CTT TGG ATT GAA AGC ATC CTA GAA CTC ATT AA - #A AAA CAC CTG CTC CCT          1909                                                                           Leu Trp Ile Glu Ser Ile Leu Glu Leu Ile Ly - #s Lys His Leu Leu Pro            #               570                                                            - CTC TGG AAT GAT GGG TGC ATC ATG GGC TTC AT - #C AGC AAG GAG CGA GAG          1957                                                                           Leu Trp Asn Asp Gly Cys Ile Met Gly Phe Il - #e Ser Lys Glu Arg Glu            #           585                                                                - CGT GCC CTG TTG AAG GAC CAG CAG CCG GGG AC - #C TTC CTG CTG CGG TTC          2005                                                                           Arg Ala Leu Leu Lys Asp Gln Gln Pro Gly Th - #r Phe Leu Leu Arg Phe            #       600                                                                    - AGT GAG AGC TCC CGG GAA GGG GCC ATC ACA TT - #C ACA TGG GTG GAG CGG          2053                                                                           Ser Glu Ser Ser Arg Glu Gly Ala Ile Thr Ph - #e Thr Trp Val Glu Arg            #   615                                                                        - TCC CAG AAC GGA GGC GAA CCT GAC TTC CAT GC - #G GTT GAA CCC TAC ACG          2101                                                                           Ser Gln Asn Gly Gly Glu Pro Asp Phe His Al - #a Val Glu Pro Tyr Thr            620                 6 - #25                 6 - #30                 6 -        #35                                                                            - AAG AAA GAA CTT TCT GCT GTT ACT TTC CCT GA - #C ATC ATT CGC AAT TAC          2149                                                                           Lys Lys Glu Leu Ser Ala Val Thr Phe Pro As - #p Ile Ile Arg Asn Tyr            #               650                                                            - AAA GTC ATG GCT GCT GAG AAT ATT CCT GAG AA - #T CCC CTG AAG TAT CTG          2197                                                                           Lys Val Met Ala Ala Glu Asn Ile Pro Glu As - #n Pro Leu Lys Tyr Leu            #           665                                                                - TAT CCA AAT ATT GAC AAA GAC CAT GCC TTT GG - #A AAG TAT TAC TCC AGG          2245                                                                           Tyr Pro Asn Ile Asp Lys Asp His Ala Phe Gl - #y Lys Tyr Tyr Ser Arg            #       680                                                                    - CCA AAG GAA GCA CCA GAG CCA ATG GAA CTT GA - #T GGC CCT AAA GGA ACT          2293                                                                           Pro Lys Glu Ala Pro Glu Pro Met Glu Leu As - #p Gly Pro Lys Gly Thr            #   695                                                                        - GGA TAT ATC AAG ACT GAG TTG ATT TCT GTG TC - #T GAA GTG TAAGTGAACA           2342                                                                           Gly Tyr Ile Lys Thr Glu Leu Ile Ser Val Se - #r Glu Val                        700                 7 - #05                 7 - #10                            - CAGAAGAGTG ACATGTTTAC AAACCTCAAG CCAGCCTTGC TCCTGGCTGG GG - #CCTGTTGA        2402                                                                           - AGATGCTTGT ATTTTACTTT TCCATTGTAA TTGCTATCGC CATCACAGCT GA - #ACTTGTTG        2462                                                                           - AGATCCCCGT GTTACTGCCT ATCAGCATTT TACTACTTTA AAAAAAAAAA AA - #AAAGCCAA        2522                                                                           - AAACCAAATT TGTATTTAAG GTATATAAAT TTTCCCAAAA CTGATACCCT TT - #GAAAAAGT        2582                                                                           #             2607 AAAG TTGAA                                                  - (2) INFORMATION FOR SEQ ID NO:6:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 712 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                  - Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu As - #p Ser Lys Phe Leu Glu          #                 15                                                           - Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pr - #o Met Glu Ile Arg Gln          #             30                                                               - Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Tr - #p Glu His Ala Ala Asn          #         45                                                                   - Asp Val Ser Phe Ala Thr Ile Arg Phe His As - #p Leu Leu Ser Gln Leu          #     60                                                                       - Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu As - #n Asn Phe Leu Leu Gln          # 80                                                                           - His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gl - #n Asp Asn Phe Gln Glu          #                 95                                                           - Asp Pro Ile Gln Met Ser Met Ile Ile Tyr Se - #r Cys Leu Lys Glu Glu          #           110                                                                - Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe As - #n Gln Ala Gln Ser Gly          #       125                                                                    - Asn Ile Gln Ser Thr Val Met Leu Asp Lys Gl - #n Lys Glu Leu Asp Ser          #   140                                                                        - Lys Val Arg Asn Val Lys Asp Lys Val Met Cy - #s Ile Glu His Glu Ile          145                 1 - #50                 1 - #55                 1 -        #60                                                                            - Lys Ser Leu Glu Asp Leu Gln Asp Glu Tyr As - #p Phe Lys Cys Lys Thr          #               175                                                            - Leu Gln Asn Arg Glu His Glu Thr Asn Gly Va - #l Ala Lys Ser Asp Gln          #           190                                                                - Lys Gln Glu Gln Leu Leu Leu Lys Lys Met Ty - #r Leu Met Leu Asp Asn          #       205                                                                    - Lys Arg Lys Glu Val Val His Lys Ile Ile Gl - #u Leu Leu Asn Val Thr          #   220                                                                        - Glu Leu Thr Gln Asn Ala Leu Ile Asn Asp Gl - #u Leu Val Glu Trp Lys          225                 2 - #30                 2 - #35                 2 -        #40                                                                            - Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pr - #o Pro Asn Ala Cys Leu          #               255                                                            - Asp Gln Leu Gln Asn Trp Phe Thr Ile Val Al - #a Glu Ser Leu Gln Gln          #           270                                                                - Val Arg Gln Gln Leu Lys Lys Leu Glu Glu Le - #u Glu Gln Lys Tyr Thr          #       285                                                                    - Tyr Glu His Asp Pro Ile Thr Lys Asn Lys Gl - #n Val Leu Trp Asp Arg          #   300                                                                        - Thr Phe Ser Leu Phe Gln Gln Leu Ile Gln Se - #r Ser Phe Val Val Glu          305                 3 - #10                 3 - #15                 3 -        #20                                                                            - Arg Gln Pro Cys Met Pro Thr His Pro Gln Ar - #g Pro Leu Val Leu Lys          #               335                                                            - Thr Gly Val Gln Phe Thr Val Lys Leu Arg Le - #u Leu Val Lys Leu Gln          #           350                                                                - Glu Leu Asn Tyr Asn Leu Lys Val Lys Val Le - #u Phe Asp Lys Asp Val          #       365                                                                    - Asn Glu Arg Asn Thr Val Lys Gly Phe Arg Ly - #s Phe Asn Ile Leu Gly          #   380                                                                        - Thr His Thr Lys Val Met Asn Met Glu Glu Se - #r Thr Asn Gly Ser Leu          385                 3 - #90                 3 - #95                 4 -        #00                                                                            - Ala Ala Glu Phe Arg His Leu Gln Leu Lys Gl - #u Gln Lys Asn Ala Gly          #               415                                                            - Thr Arg Thr Asn Glu Gly Pro Leu Ile Val Th - #r Glu Glu Leu His Ser          #           430                                                                - Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gl - #y Leu Val Ile Asp Leu          #       445                                                                    - Glu Thr Thr Ser Leu Pro Val Val Val Ile Se - #r Asn Val Ser Gln Leu          #   460                                                                        - Pro Ser Gly Trp Ala Ser Ile Leu Trp Tyr As - #n Met Leu Val Ala Glu          465                 4 - #70                 4 - #75                 4 -        #80                                                                            - Pro Arg Asn Leu Ser Phe Phe Leu Thr Pro Pr - #o Cys Ala Arg Trp Ala          #               495                                                            - Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Se - #r Ser Val Thr Lys Arg          #           510                                                                - Gly Leu Asn Val Asp Gln Leu Asn Met Leu Gl - #y Glu Lys Leu Leu Gly          #       525                                                                    - Pro Asn Ala Ser Pro Asp Gly Leu Ile Pro Tr - #p Thr Arg Phe Cys Lys          #   540                                                                        - Glu Asn Ile Asn Asp Lys Asn Phe Pro Phe Tr - #p Leu Trp Ile Glu Ser          545                 5 - #50                 5 - #55                 5 -        #60                                                                            - Ile Leu Glu Leu Ile Lys Lys His Leu Leu Pr - #o Leu Trp Asn Asp Gly          #               575                                                            - Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Gl - #u Arg Ala Leu Leu Lys          #           590                                                                - Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Ph - #e Ser Glu Ser Ser Arg          #       605                                                                    - Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Ar - #g Ser Gln Asn Gly Gly          #   620                                                                        - Glu Pro Asp Phe His Ala Val Glu Pro Tyr Th - #r Lys Lys Glu Leu Ser          625                 6 - #30                 6 - #35                 6 -        #40                                                                            - Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Ty - #r Lys Val Met Ala Ala          #               655                                                            - Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Le - #u Tyr Pro Asn Ile Asp          #           670                                                                - Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Ar - #g Pro Lys Glu Ala Pro          #       685                                                                    - Glu Pro Met Glu Leu Asp Gly Pro Lys Gly Th - #r Gly Tyr Ile Lys Thr          #   700                                                                        - Glu Leu Ile Ser Val Ser Glu Val                                              705                 7 - #10                                                    - (2) INFORMATION FOR SEQ ID NO:7:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 2277 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: unknown                                                -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Mouse                                                  -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: Murine Stat9 - #1                                         -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION: 5..2251                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                  - CAGG ATG TCA CAG TGG TTC GAG CTT CAG CAG C - #TG GAC TCC AAG TTC CTG           49                                                                                Met Ser Gln Trp Phe Glu Leu Gln - # Gln Leu Asp Ser Lys Phe Leu           #    15                                                                        - GAG CAG GTC CAC CAG CTG TAC GAT GAC AGT TT - #C CCC ATG GAA ATC AGA            97                                                                           Glu Gln Val His Gln Leu Tyr Asp Asp Ser Ph - #e Pro Met Glu Ile Arg            #                 30                                                           - CAG TAC CTG GCC CAG TGG CTG GAA AAG CAA GA - #C TGG GAG CAC GCT GCC           145                                                                           Gln Tyr Leu Ala Gln Trp Leu Glu Lys Gln As - #p Trp Glu His Ala Ala            #             45                                                               - TAT GAT GTC TCG TTT GCG ACC ATC CGC TTC CA - #T GAC CTC CTC TCA CAG           193                                                                           Tyr Asp Val Ser Phe Ala Thr Ile Arg Phe Hi - #s Asp Leu Leu Ser Gln            #         60                                                                   - CTG GAC GAC CAG TAC AGC CGC TTT TCT CTG GA - #G AAT AAT TTC TTG TTG           241                                                                           Leu Asp Asp Gln Tyr Ser Arg Phe Ser Leu Gl - #u Asn Asn Phe Leu Leu            #     75                                                                       - CAG CAC AAC ATA CGG AAA AGC AAG CGT AAT CT - #C CAG GAT AAC TTC CAA           289                                                                           Gln His Asn Ile Arg Lys Ser Lys Arg Asn Le - #u Gln Asp Asn Phe Gln            # 95                                                                           - GAA GAT CCC GTA CAG ATG TCC ATG ATC ATC TA - #C AAC TGT CTG AAG GAA           337                                                                           Glu Asp Pro Val Gln Met Ser Met Ile Ile Ty - #r Asn Cys Leu Lys Glu            #               110                                                            - GAA AGG AAG ATT TTG GAA AAT GCC CAA AGA TT - #T AAT CAG GCC CAG GAG           385                                                                           Glu Arg Lys Ile Leu Glu Asn Ala Gln Arg Ph - #e Asn Gln Ala Gln Glu            #           125                                                                - GGA AAT ATT CAG AAC ACT GTG ATG TTA GAT AA - #A CAG AAG GAG CTG GAC           433                                                                           Gly Asn Ile Gln Asn Thr Val Met Leu Asp Ly - #s Gln Lys Glu Leu Asp            #       140                                                                    - AGT AAA GTC AGA AAT GTG AAG GAT CAA GTC AT - #G TGC ATA GAG CAG GAA           481                                                                           Ser Lys Val Arg Asn Val Lys Asp Gln Val Me - #t Cys Ile Glu Gln Glu            #   155                                                                        - ATC AAG ACC CTA GAA GAA TTA CAA GAT GAA TA - #T GAC TTT AAA TGC AAA           529                                                                           Ile Lys Thr Leu Glu Glu Leu Gln Asp Glu Ty - #r Asp Phe Lys Cys Lys            160                 1 - #65                 1 - #70                 1 -        #75                                                                            - ACC TCT CAG AAC AGA GAA GGT GAA GCC AAT GG - #T GTG GCG AAG AGC GAC           577                                                                           Thr Ser Gln Asn Arg Glu Gly Glu Ala Asn Gl - #y Val Ala Lys Ser Asp            #               190                                                            - CAA AAA CAG GAA CAG CTG CTG CTC CAC AAG AT - #G TTT TTA ATG CTT GAC           625                                                                           Gln Lys Gln Glu Gln Leu Leu Leu His Lys Me - #t Phe Leu Met Leu Asp            #           205                                                                - AAT AAG AGA AAG GAG ATA ATT CAC AAA ATC AG - #A GAG TTG CTG AAT TCC           673                                                                           Asn Lys Arg Lys Glu Ile Ile His Lys Ile Ar - #g Glu Leu Leu Asn Ser            #       220                                                                    - ATC GAG CTC ACT CAG AAC ACT CTG ATT AAT GA - #C GAG CTC GTG GAG TGG           721                                                                           Ile Glu Leu Thr Gln Asn Thr Leu Ile Asn As - #p Glu Leu Val Glu Trp            #   235                                                                        - AAG CGA AGG CAG CAG AGC GCC TGC ATC GGG GG - #A CCG CCC AAC GCC TGC           769                                                                           Lys Arg Arg Gln Gln Ser Ala Cys Ile Gly Gl - #y Pro Pro Asn Ala Cys            240                 2 - #45                 2 - #50                 2 -        #55                                                                            - CTG GAT CAG CTG CAA ACG TGG TTC ACC ATT GT - #T GCA GAG ACC CTG CAG           817                                                                           Leu Asp Gln Leu Gln Thr Trp Phe Thr Ile Va - #l Ala Glu Thr Leu Gln            #               270                                                            - CAG ATC CGT CAG CAG CTT AAA AAG CTG GAG GA - #G TTG GAA CAG AAA TTC           865                                                                           Gln Ile Arg Gln Gln Leu Lys Lys Leu Glu Gl - #u Leu Glu Gln Lys Phe            #           285                                                                - ACC TAT GAG CCC GAC CCT ATT ACA AAA AAC AA - #G CAG GTG TTG TCA GAT           913                                                                           Thr Tyr Glu Pro Asp Pro Ile Thr Lys Asn Ly - #s Gln Val Leu Ser Asp            #       300                                                                    - CGA ACC TTC CTC CTC TTC CAG CAG CTC ATT CA - #G AGC TCC TTC GTG GTA           961                                                                           Arg Thr Phe Leu Leu Phe Gln Gln Leu Ile Gl - #n Ser Ser Phe Val Val            #   315                                                                        - GAA CGA CAG CCG TGC ATG CCC ACT CAC CCG CA - #G AGG CCC CTG GTC TTG          1009                                                                           Glu Arg Gln Pro Cys Met Pro Thr His Pro Gl - #n Arg Pro Leu Val Leu            320                 3 - #25                 3 - #30                 3 -        #35                                                                            - AAG ACT GGG GTA CAG TTC ACT GTC AAG TCG AG - #A CTG TTG GTG AAA TTG          1057                                                                           Lys Thr Gly Val Gln Phe Thr Val Lys Ser Ar - #g Leu Leu Val Lys Leu            #               350                                                            - CAA GAG TCG AAT CTA TTA ACG AAA GTG AAA TG - #T CAC TTT GAC AAA GAT          1105                                                                           Gln Glu Ser Asn Leu Leu Thr Lys Val Lys Cy - #s His Phe Asp Lys Asp            #           365                                                                - GTG AAC GAG AAA AAC ACA GTT AAA GGA TTT CG - #G AAG TTC AAC ATC TTG          1153                                                                           Val Asn Glu Lys Asn Thr Val Lys Gly Phe Ar - #g Lys Phe Asn Ile Leu            #       380                                                                    - GGT ACG CAC ACA AAA GTG ATG AAC ATG GAA GA - #A TCC ACC AAC GGA AGT          1201                                                                           Gly Thr His Thr Lys Val Met Asn Met Glu Gl - #u Ser Thr Asn Gly Ser            #   395                                                                        - CTG GCA GCT GAG CTC CGA CAC CTG CAA CTG AA - #G GAA CAG AAA AAC GCT          1249                                                                           Leu Ala Ala Glu Leu Arg His Leu Gln Leu Ly - #s Glu Gln Lys Asn Ala            400                 4 - #05                 4 - #10                 4 -        #15                                                                            - GGG AAC AGA ACT AAT GAG GGG CCT CTC ATT GT - #C ACC GAA GAA CTT CAC          1297                                                                           Gly Asn Arg Thr Asn Glu Gly Pro Leu Ile Va - #l Thr Glu Glu Leu His            #               430                                                            - TCT CTT AGC TTT GAA ACC CAG TTG TGC CAG CC - #A GGC TTG GTG ATT GAC          1345                                                                           Ser Leu Ser Phe Glu Thr Gln Leu Cys Gln Pr - #o Gly Leu Val Ile Asp            #           445                                                                - CTG GAG ACC ACC TCT CTT CCT GTC GTG GTG AT - #C TCC AAC GTC AGC CAG          1393                                                                           Leu Glu Thr Thr Ser Leu Pro Val Val Val Il - #e Ser Asn Val Ser Gln            #       460                                                                    - CTC CCC AGT GGC TGG GCG TCT ATC CTG TGG TA - #C AAC ATG CTG GTG ACA          1441                                                                           Leu Pro Ser Gly Trp Ala Ser Ile Leu Trp Ty - #r Asn Met Leu Val Thr            #   475                                                                        - GAG CCC AGG AAT CTC TCC TTC TTC CTG AAC CC - #C CCG TGC GCG TGG TGG          1489                                                                           Glu Pro Arg Asn Leu Ser Phe Phe Leu Asn Pr - #o Pro Cys Ala Trp Trp            480                 4 - #85                 4 - #90                 4 -        #95                                                                            - TCC CAG CTC TCA GAG GTG TTG AGT TGG CAG TT - #T TCA TCA GTC ACC AAG          1537                                                                           Ser Gln Leu Ser Glu Val Leu Ser Trp Gln Ph - #e Ser Ser Val Thr Lys            #               510                                                            - AGA GGT CTG AAC GCA GAC CAG CTG AGC ATG CT - #G GGA GAG AAG CTG CTG          1585                                                                           Arg Gly Leu Asn Ala Asp Gln Leu Ser Met Le - #u Gly Glu Lys Leu Leu            #           525                                                                - GGC CCT AAT GCT GGC CCT GAT GGT CTT ATT CC - #A TGG ACA AGG TTT TGT          1633                                                                           Gly Pro Asn Ala Gly Pro Asp Gly Leu Ile Pr - #o Trp Thr Arg Phe Cys            #       540                                                                    - AAG GAA AAT ATT AAT GAT AAA AAT TTC TCC TT - #C TGG CCT TGG ATT GAC          1681                                                                           Lys Glu Asn Ile Asn Asp Lys Asn Phe Ser Ph - #e Trp Pro Trp Ile Asp            #   555                                                                        - ACC ATC CTA GAG CTC ATT AAG AAC GAC CTG CT - #G TGC CTC TGG AAT GAT          1729                                                                           Thr Ile Leu Glu Leu Ile Lys Asn Asp Leu Le - #u Cys Leu Trp Asn Asp            560                 5 - #65                 5 - #70                 5 -        #75                                                                            - GGG TGC ATT ATG GGC TTC ATC AGC AAG GAG CG - #A GAA CGC GCT CTG CTC          1777                                                                           Gly Cys Ile Met Gly Phe Ile Ser Lys Glu Ar - #g Glu Arg Ala Leu Leu            #               590                                                            - AAG GAC CAG CAG CCA GGG ACG TTC CTG CTT AG - #A TTC AGT GAG AGC TCC          1825                                                                           Lys Asp Gln Gln Pro Gly Thr Phe Leu Leu Ar - #g Phe Ser Glu Ser Ser            #           605                                                                - CGG GAA GGG GCC ATC ACA TTC ACA TGG GTG GA - #A CGG TCC CAG AAC GGA          1873                                                                           Arg Glu Gly Ala Ile Thr Phe Thr Trp Val Gl - #u Arg Ser Gln Asn Gly            #       620                                                                    - GGT GAA CCT GAC TTC CAT GCC GTG GAG CCC TA - #C ACG AAA AAA GAA CTT          1921                                                                           Gly Glu Pro Asp Phe His Ala Val Glu Pro Ty - #r Thr Lys Lys Glu Leu            #   635                                                                        - TCA GCT GTT ACT TTC CCA GAT ATT ATT CGC AA - #C TAC AAA GTC ATG GCT          1969                                                                           Ser Ala Val Thr Phe Pro Asp Ile Ile Arg As - #n Tyr Lys Val Met Ala            640                 6 - #45                 6 - #50                 6 -        #55                                                                            - GCC GAG AAC ATA CCA GAG AAT CCC CTG AAG TA - #T CTG TAC CCC AAT ATT          2017                                                                           Ala Glu Asn Ile Pro Glu Asn Pro Leu Lys Ty - #r Leu Tyr Pro Asn Ile            #               670                                                            - GAC AAA GAC CAC GCC TTT GGG AAG TAT TAT TC - #C AGA CCA AAG GAA GCA          2065                                                                           Asp Lys Asp His Ala Phe Gly Lys Tyr Tyr Se - #r Arg Pro Lys Glu Ala            #           685                                                                - CCA GAA CCG ATG GAG CTT GAC GAC CCT AAG CG - #A ACT GGA TAC ATC AAG          2113                                                                           Pro Glu Pro Met Glu Leu Asp Asp Pro Lys Ar - #g Thr Gly Tyr Ile Lys            #       700                                                                    - ACT GAG TTG ATT TCT GTG TCT GAA GTC CAC CC - #T TCT AGA CTT CAG ACC          2161                                                                           Thr Glu Leu Ile Ser Val Ser Glu Val His Pr - #o Ser Arg Leu Gln Thr            #   715                                                                        - ACA GAC AAC CTG CTT CCC ATG TCT CCA GAG GA - #G TTT GAT GAG ATG TCC          2209                                                                           Thr Asp Asn Leu Leu Pro Met Ser Pro Glu Gl - #u Phe Asp Glu Met Ser            720                 7 - #25                 7 - #30                 7 -        #35                                                                            - CGG ATA GTG GGC CCC GAA TTT GAC AGT ATG AT - #G AGC ACA GTA                  #2251                                                                          Arg Ile Val Gly Pro Glu Phe Asp Ser Met Me - #t Ser Thr Val                    #               745                                                            #            2277  TCTG GCGACA                                                 - (2) INFORMATION FOR SEQ ID NO:8:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 749 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                  - Met Ser Gln Trp Phe Glu Leu Gln Gln Leu As - #p Ser Lys Phe Leu Glu          #                 15                                                           - Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pr - #o Met Glu Ile Arg Gln          #             30                                                               - Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Tr - #p Glu His Ala Ala Tyr          #         45                                                                   - Asp Val Ser Phe Ala Thr Ile Arg Phe His As - #p Leu Leu Ser Gln Leu          #     60                                                                       - Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu As - #n Asn Phe Leu Leu Gln          # 80                                                                           - His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gl - #n Asp Asn Phe Gln Glu          #                 95                                                           - Asp Pro Val Gln Met Ser Met Ile Ile Tyr As - #n Cys Leu Lys Glu Glu          #           110                                                                - Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe As - #n Gln Ala Gln Glu Gly          #       125                                                                    - Asn Ile Gln Asn Thr Val Met Leu Asp Lys Gl - #n Lys Glu Leu Asp Ser          #   140                                                                        - Lys Val Arg Asn Val Lys Asp Gln Val Met Cy - #s Ile Glu Gln Glu Ile          145                 1 - #50                 1 - #55                 1 -        #60                                                                            - Lys Thr Leu Glu Glu Leu Gln Asp Glu Tyr As - #p Phe Lys Cys Lys Thr          #               175                                                            - Ser Gln Asn Arg Glu Gly Glu Ala Asn Gly Va - #l Ala Lys Ser Asp Gln          #           190                                                                - Lys Gln Glu Gln Leu Leu Leu His Lys Met Ph - #e Leu Met Leu Asp Asn          #       205                                                                    - Lys Arg Lys Glu Ile Ile His Lys Ile Arg Gl - #u Leu Leu Asn Ser Ile          #   220                                                                        - Glu Leu Thr Gln Asn Thr Leu Ile Asn Asp Gl - #u Leu Val Glu Trp Lys          225                 2 - #30                 2 - #35                 2 -        #40                                                                            - Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pr - #o Pro Asn Ala Cys Leu          #               255                                                            - Asp Gln Leu Gln Thr Trp Phe Thr Ile Val Al - #a Glu Thr Leu Gln Gln          #           270                                                                - Ile Arg Gln Gln Leu Lys Lys Leu Glu Glu Le - #u Glu Gln Lys Phe Thr          #       285                                                                    - Tyr Glu Pro Asp Pro Ile Thr Lys Asn Lys Gl - #n Val Leu Ser Asp Arg          #   300                                                                        - Thr Phe Leu Leu Phe Gln Gln Leu Ile Gln Se - #r Ser Phe Val Val Glu          305                 3 - #10                 3 - #15                 3 -        #20                                                                            - Arg Gln Pro Cys Met Pro Thr His Pro Gln Ar - #g Pro Leu Val Leu Lys          #               335                                                            - Thr Gly Val Gln Phe Thr Val Lys Ser Arg Le - #u Leu Val Lys Leu Gln          #           350                                                                - Glu Ser Asn Leu Leu Thr Lys Val Lys Cys Hi - #s Phe Asp Lys Asp Val          #       365                                                                    - Asn Glu Lys Asn Thr Val Lys Gly Phe Arg Ly - #s Phe Asn Ile Leu Gly          #   380                                                                        - Thr His Thr Lys Val Met Asn Met Glu Glu Se - #r Thr Asn Gly Ser Leu          385                 3 - #90                 3 - #95                 4 -        #00                                                                            - Ala Ala Glu Leu Arg His Leu Gln Leu Lys Gl - #u Gln Lys Asn Ala Gly          #               415                                                            - Asn Arg Thr Asn Glu Gly Pro Leu Ile Val Th - #r Glu Glu Leu His Ser          #           430                                                                - Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gl - #y Leu Val Ile Asp Leu          #       445                                                                    - Glu Thr Thr Ser Leu Pro Val Val Val Ile Se - #r Asn Val Ser Gln Leu          #   460                                                                        - Pro Ser Gly Trp Ala Ser Ile Leu Trp Tyr As - #n Met Leu Val Thr Glu          465                 4 - #70                 4 - #75                 4 -        #80                                                                            - Pro Arg Asn Leu Ser Phe Phe Leu Asn Pro Pr - #o Cys Ala Trp Trp Ser          #               495                                                            - Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Se - #r Ser Val Thr Lys Arg          #           510                                                                - Gly Leu Asn Ala Asp Gln Leu Ser Met Leu Gl - #y Glu Lys Leu Leu Gly          #       525                                                                    - Pro Asn Ala Gly Pro Asp Gly Leu Ile Pro Tr - #p Thr Arg Phe Cys Lys          #   540                                                                        - Glu Asn Ile Asn Asp Lys Asn Phe Ser Phe Tr - #p Pro Trp Ile Asp Thr          545                 5 - #50                 5 - #55                 5 -        #60                                                                            - Ile Leu Glu Leu Ile Lys Asn Asp Leu Leu Cy - #s Leu Trp Asn Asp Gly          #               575                                                            - Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Gl - #u Arg Ala Leu Leu Lys          #           590                                                                - Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Ph - #e Ser Glu Ser Ser Arg          #       605                                                                    - Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Ar - #g Ser Gln Asn Gly Gly          #   620                                                                        - Glu Pro Asp Phe His Ala Val Glu Pro Tyr Th - #r Lys Lys Glu Leu Ser          625                 6 - #30                 6 - #35                 6 -        #40                                                                            - Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Ty - #r Lys Val Met Ala Ala          #               655                                                            - Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Le - #u Tyr Pro Asn Ile Asp          #           670                                                                - Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Ar - #g Pro Lys Glu Ala Pro          #       685                                                                    - Glu Pro Met Glu Leu Asp Asp Pro Lys Arg Th - #r Gly Tyr Ile Lys Thr          #   700                                                                        - Glu Leu Ile Ser Val Ser Glu Val His Pro Se - #r Arg Leu Gln Thr Thr          705                 7 - #10                 7 - #15                 7 -        #20                                                                            - Asp Asn Leu Leu Pro Met Ser Pro Glu Glu Ph - #e Asp Glu Met Ser Arg          #               735                                                            - Ile Val Gly Pro Glu Phe Asp Ser Met Met Se - #r Thr Val                      #           745                                                                - (2) INFORMATION FOR SEQ ID NO:9:                                             -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 2375 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: unknown                                                -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Mouse                                                  -    (vii) IMMEDIATE SOURCE:                                                             (A) LIBRARY: splenic/thymi - #c                                                (B) CLONE: Murine 13sf1                                              -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION: 34..2277                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                  - TGCCACTACC TGGACGGAGA GAGAGAGAGC AGC ATG TCT CAG TG - #G AAT CAA GTC           54                                                                           # Met Ser Gln Trp Asn Gln Val                                                  #5  1                                                                          - CAA CAA TTA GAA ATC AAG TTT TTG GAG CAA GT - #A GAT CAG TTC TAT GAT           102                                                                           Gln Gln Leu Glu Ile Lys Phe Leu Glu Gln Va - #l Asp Gln Phe Tyr Asp            #         20                                                                   - GAC AAC TTT CCT ATG GAA ATC CGG CAT CTG CT - #A GCT CAG TGG ATT GAG           150                                                                           Asp Asn Phe Pro Met Glu Ile Arg His Leu Le - #u Ala Gln Trp Ile Glu            #     35                                                                       - ACT CAA GAC TGG GAA GTA GCT TCT AAC AAT GA - #A ACT ATG GCA ACA ATT           198                                                                           Thr Gln Asp Trp Glu Val Ala Ser Asn Asn Gl - #u Thr Met Ala Thr Ile            # 55                                                                           - CTG CTT CAA AAC TTA CTA ATA CAA TTG GAT GA - #A CAG TTG GGG CGG GTT           246                                                                           Leu Leu Gln Asn Leu Leu Ile Gln Leu Asp Gl - #u Gln Leu Gly Arg Val            #                 70                                                           - TCC AAA GAA AAA AAT CTG CTA TTG ATT CAC AA - #T CTA AAG AGA ATT AGA           294                                                                           Ser Lys Glu Lys Asn Leu Leu Leu Ile His As - #n Leu Lys Arg Ile Arg            #             85                                                               - AAA GTT CTT CAG GGC AAG TTT CAT GGA AAT CC - #A ATG CAT GTA GCT GTG           342                                                                           Lys Val Leu Gln Gly Lys Phe His Gly Asn Pr - #o Met His Val Ala Val            #        100                                                                   - GTA ATT TCA AAT TGC TTA AGG GAA GAG AGG AG - #A ATA TTG GCT GCA GCC           390                                                                           Val Ile Ser Asn Cys Leu Arg Glu Glu Arg Ar - #g Ile Leu Ala Ala Ala            #   115                                                                        - AAC ATG CCT ATC CAG GGA CCT CTG GAG AAA TC - #C TTA CAG AGT TCT TCA           438                                                                           Asn Met Pro Ile Gln Gly Pro Leu Glu Lys Se - #r Leu Gln Ser Ser Ser            120                 1 - #25                 1 - #30                 1 -        #35                                                                            - GTT TCT GAA AGA CAA AGG AAT GTG GAA CAC AA - #A GTG TCT GCC ATT AAA           486                                                                           Val Ser Glu Arg Gln Arg Asn Val Glu His Ly - #s Val Ser Ala Ile Lys            #               150                                                            - AAC AGT GTG CAG ATG ACA GAA CAA GAT ACC AA - #A TAC TTA GAA GAC CTG           534                                                                           Asn Ser Val Gln Met Thr Glu Gln Asp Thr Ly - #s Tyr Leu Glu Asp Leu            #           165                                                                - CAA GAT GAG TTT GAC TAC AGG TAT AAA ACA AT - #T CAG ACA ATG GAT CAG           582                                                                           Gln Asp Glu Phe Asp Tyr Arg Tyr Lys Thr Il - #e Gln Thr Met Asp Gln            #       180                                                                    - GGT GAC AAA AAC AGT ATC CTG GTG AAC CAG GA - #A GTT TTG ACA CTG CTG           630                                                                           Gly Asp Lys Asn Ser Ile Leu Val Asn Gln Gl - #u Val Leu Thr Leu Leu            #   195                                                                        - CAA GAA ATG CTT AAT AGT CTG GAC TTC AAG AG - #A AAG GAA GCA CTC AGT           678                                                                           Gln Glu Met Leu Asn Ser Leu Asp Phe Lys Ar - #g Lys Glu Ala Leu Ser            200                 2 - #05                 2 - #10                 2 -        #15                                                                            - AAG ATG ACG CAG ATA GTG AAC GAG ACA GAC CT - #G CTC ATG AAC AGC ATG           726                                                                           Lys Met Thr Gln Ile Val Asn Glu Thr Asp Le - #u Leu Met Asn Ser Met            #               230                                                            - CTT CTA GAA GAG CTG CAG GAC TGG AAA AAG CG - #G CAC AGG ATT GCC TGC           774                                                                           Leu Leu Glu Glu Leu Gln Asp Trp Lys Lys Ar - #g His Arg Ile Ala Cys            #           245                                                                - ATT GGT GGC CCG CTC CAC AAT GGG CTG GAC CA - #G CTT CAG AAC TGC TTT           822                                                                           Ile Gly Gly Pro Leu His Asn Gly Leu Asp Gl - #n Leu Gln Asn Cys Phe            #       260                                                                    - ACC CTA CTG GCA GAG AGT CTT TTC CAA CTC AG - #A CAG CAA CTG GAG AAA           870                                                                           Thr Leu Leu Ala Glu Ser Leu Phe Gln Leu Ar - #g Gln Gln Leu Glu Lys            #   275                                                                        - CTA CAG GAG CAA TCT ACT AAA ATG ACC TAT GA - #A GGG GAT CCC ATC CCT           918                                                                           Leu Gln Glu Gln Ser Thr Lys Met Thr Tyr Gl - #u Gly Asp Pro Ile Pro            280                 2 - #85                 2 - #90                 2 -        #95                                                                            - GCT CAA AGA GCA CAC CTC CTG GAA AGA GCT AC - #C TTC CTG ATC TAC AAC           966                                                                           Ala Gln Arg Ala His Leu Leu Glu Arg Ala Th - #r Phe Leu Ile Tyr Asn            #               310                                                            - CTT TTC AAG AAC TCA TTT GTG GTC GAG CGA CA - #C GCA TGC ATG CCA ACG          1014                                                                           Leu Phe Lys Asn Ser Phe Val Val Glu Arg Hi - #s Ala Cys Met Pro Thr            #           325                                                                - CAC CCT CAG AGG CCG ATG GTA CTT AAA ACC CT - #C ATT CAG TTC ACT GTA          1062                                                                           His Pro Gln Arg Pro Met Val Leu Lys Thr Le - #u Ile Gln Phe Thr Val            #       340                                                                    - AAA CTG AGA TTA CTA ATA AAA TTG CCG GAA CT - #A AAC TAT CAG GTG AAA          1110                                                                           Lys Leu Arg Leu Leu Ile Lys Leu Pro Glu Le - #u Asn Tyr Gln Val Lys            #   355                                                                        - GTA AAG GCG TCC ATT GAC AAG AAT GTT TCA AC - #T CTA AGC AAT AGA AGA          1158                                                                           Val Lys Ala Ser Ile Asp Lys Asn Val Ser Th - #r Leu Ser Asn Arg Arg            360                 3 - #65                 3 - #70                 3 -        #75                                                                            - TTT GTG CTT TGT GGA ACT CAC GTC AAA GCT AT - #G TCC AGT GAG GAA TCT          1206                                                                           Phe Val Leu Cys Gly Thr His Val Lys Ala Me - #t Ser Ser Glu Glu Ser            #               390                                                            - TCC AAT GGG AGC CTC TCA GTG GAG TTA GAC AT - #T GCA ACC CAA GGA GAT          1254                                                                           Ser Asn Gly Ser Leu Ser Val Glu Leu Asp Il - #e Ala Thr Gln Gly Asp            #           405                                                                - GAA GTG CAG TAC TGG AGT AAA GGA AAC GAG GG - #C TGC CAC ATG GTG ACA          1302                                                                           Glu Val Gln Tyr Trp Ser Lys Gly Asn Glu Gl - #y Cys His Met Val Thr            #       420                                                                    - GAG GAG TTG CAT TCC ATA ACC TTT GAG ACC CA - #G ATC TGC CTC TAT GGC          1350                                                                           Glu Glu Leu His Ser Ile Thr Phe Glu Thr Gl - #n Ile Cys Leu Tyr Gly            #   435                                                                        - CTC ACC ATT AAC CTA GAG ACC AGC TCA TTA CC - #T GTC GTG ATG ATT TCT          1398                                                                           Leu Thr Ile Asn Leu Glu Thr Ser Ser Leu Pr - #o Val Val Met Ile Ser            440                 4 - #45                 4 - #50                 4 -        #55                                                                            - AAT GTC AGC CAA CTA CCT AAT GCA TGG GCA TC - #C ATC ATT TGG TAC AAT          1446                                                                           Asn Val Ser Gln Leu Pro Asn Ala Trp Ala Se - #r Ile Ile Trp Tyr Asn            #               470                                                            - GTA TCA ACT AAC GAC TCC CAG AAC TTG GTT TT - #C TTT AAT AAC CCT CCA          1494                                                                           Val Ser Thr Asn Asp Ser Gln Asn Leu Val Ph - #e Phe Asn Asn Pro Pro            #           485                                                                - TCT GTC ACT TTG GGC CAA CTC CTG GAA GTG AT - #G AGC TGG CAA TTT TCA          1542                                                                           Ser Val Thr Leu Gly Gln Leu Leu Glu Val Me - #t Ser Trp Gln Phe Ser            #       500                                                                    - TCC TAT GTC GGT CGT GGC CTT AAT TCA GAG CA - #G CTC AAC ATG CTG GCA          1590                                                                           Ser Tyr Val Gly Arg Gly Leu Asn Ser Glu Gl - #n Leu Asn Met Leu Ala            #   515                                                                        - GAG AAG CTC ACA GTT CAG TCT AAC TAC AAT GA - #T GGT CAC CTC ACC TGG          1638                                                                           Glu Lys Leu Thr Val Gln Ser Asn Tyr Asn As - #p Gly His Leu Thr Trp            520                 5 - #25                 5 - #30                 5 -        #35                                                                            - GCC AAG TTC TGC AAG GAA CAT TTG CCT GGC AA - #A ACA TTT ACC TTC TGG          1686                                                                           Ala Lys Phe Cys Lys Glu His Leu Pro Gly Ly - #s Thr Phe Thr Phe Trp            #               550                                                            - ACT TGG CTT GAA GCA ATA TTG GAC CTA ATT AA - #A AAA CAT ATT CTT CCC          1734                                                                           Thr Trp Leu Glu Ala Ile Leu Asp Leu Ile Ly - #s Lys His Ile Leu Pro            #           565                                                                - CTC TGG ATT GAT GGG TAC ATC ATG GGA TTT GT - #T AGT AAA GAG AAG GAA          1782                                                                           Leu Trp Ile Asp Gly Tyr Ile Met Gly Phe Va - #l Ser Lys Glu Lys Glu            #       580                                                                    - CGG CTT CTG CTC AAA GAT AAA ATG CCT GGG AC - #A TTT TTG TTA AGA TTC          1830                                                                           Arg Leu Leu Leu Lys Asp Lys Met Pro Gly Th - #r Phe Leu Leu Arg Phe            #   595                                                                        - AGT GAG AGC CAT CTT GGA GGG ATA ACC TTC AC - #C TGG GTG GAC CAA TCT          1878                                                                           Ser Glu Ser His Leu Gly Gly Ile Thr Phe Th - #r Trp Val Asp Gln Ser            600                 6 - #05                 6 - #10                 6 -        #15                                                                            - GAA AAT GGA GAA GTG AGA TTC CAC TCT GTA GA - #A CCC TAC AAC AAA GGG          1926                                                                           Glu Asn Gly Glu Val Arg Phe His Ser Val Gl - #u Pro Tyr Asn Lys Gly            #               630                                                            - AGA CTG TCG GCT CTG GCC TTC GCT GAC ATC CT - #G CGA GAC TAC AAG GTT          1974                                                                           Arg Leu Ser Ala Leu Ala Phe Ala Asp Ile Le - #u Arg Asp Tyr Lys Val            #           645                                                                - ATC ATG GCT GAA AAC ATC CCT GAA AAC CCT CT - #G AAG TAC CTC TAC CCT          2022                                                                           Ile Met Ala Glu Asn Ile Pro Glu Asn Pro Le - #u Lys Tyr Leu Tyr Pro            #       660                                                                    - GAC ATT CCC AAA GAC AAA GCC TTT GGC AAA CA - #C TAC AGC TCC CAG CCG          2070                                                                           Asp Ile Pro Lys Asp Lys Ala Phe Gly Lys Hi - #s Tyr Ser Ser Gln Pro            #   675                                                                        - TGC GAA GTC TCA AGA CCA ACC GAA CGG GGA GA - #C AAG GGT TAC GTC CCC          2118                                                                           Cys Glu Val Ser Arg Pro Thr Glu Arg Gly As - #p Lys Gly Tyr Val Pro            680                 6 - #85                 6 - #90                 6 -        #95                                                                            - TCT GTT TTT ATC CCC ATT TCA ACA ATC CGA AG - #C GAT TCC ACG GAG CCA          2166                                                                           Ser Val Phe Ile Pro Ile Ser Thr Ile Arg Se - #r Asp Ser Thr Glu Pro            #               710                                                            - CAA TCT CCT TCA GAC CTT CTC CCC ATG TCT CC - #A AGT GCA TAT GCT GTG          2214                                                                           Gln Ser Pro Ser Asp Leu Leu Pro Met Ser Pr - #o Ser Ala Tyr Ala Val            #           725                                                                - CTG AGA GAA AAC CTG AGC CCA ACG ACA ATT GA - #A ACT GCA ATG AAT TCC          2262                                                                           Leu Arg Glu Asn Leu Ser Pro Thr Thr Ile Gl - #u Thr Ala Met Asn Ser            #       740                                                                    - CCA TAT TCT GCT GAA TGACGGTGCA AACGGACACT TTAAAGAAG - #G AAGCAGATGA          2317                                                                           Pro Tyr Ser Ala Glu                                                                745                                                                        - AACTGGAGAG TGTTCTTTAC CATAGATCAC AATTTATTTC TTCGGCTTTG TA - #AATACC          2375                                                                           - (2) INFORMATION FOR SEQ ID NO:10:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 748 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                 - Met Ser Gln Trp Asn Gln Val Gln Gln Leu Gl - #u Ile Lys Phe Leu Glu          #                 15                                                           - Gln Val Asp Gln Phe Tyr Asp Asp Asn Phe Pr - #o Met Glu Ile Arg His          #             30                                                               - Leu Leu Ala Gln Trp Ile Glu Thr Gln Asp Tr - #p Glu Val Ala Ser Asn          #         45                                                                   - Asn Glu Thr Met Ala Thr Ile Leu Leu Gln As - #n Leu Leu Ile Gln Leu          #     60                                                                       - Asp Glu Gln Leu Gly Arg Val Ser Lys Glu Ly - #s Asn Leu Leu Leu Ile          # 80                                                                           - His Asn Leu Lys Arg Ile Arg Lys Val Leu Gl - #n Gly Lys Phe His Gly          #                 95                                                           - Asn Pro Met His Val Ala Val Val Ile Ser As - #n Cys Leu Arg Glu Glu          #           110                                                                - Arg Arg Ile Leu Ala Ala Ala Asn Met Pro Il - #e Gln Gly Pro Leu Glu          #       125                                                                    - Lys Ser Leu Gln Ser Ser Ser Val Ser Glu Ar - #g Gln Arg Asn Val Glu          #   140                                                                        - His Lys Val Ser Ala Ile Lys Asn Ser Val Gl - #n Met Thr Glu Gln Asp          145                 1 - #50                 1 - #55                 1 -        #60                                                                            - Thr Lys Tyr Leu Glu Asp Leu Gln Asp Glu Ph - #e Asp Tyr Arg Tyr Lys          #               175                                                            - Thr Ile Gln Thr Met Asp Gln Gly Asp Lys As - #n Ser Ile Leu Val Asn          #           190                                                                - Gln Glu Val Leu Thr Leu Leu Gln Glu Met Le - #u Asn Ser Leu Asp Phe          #       205                                                                    - Lys Arg Lys Glu Ala Leu Ser Lys Met Thr Gl - #n Ile Val Asn Glu Thr          #   220                                                                        - Asp Leu Leu Met Asn Ser Met Leu Leu Glu Gl - #u Leu Gln Asp Trp Lys          225                 2 - #30                 2 - #35                 2 -        #40                                                                            - Lys Arg His Arg Ile Ala Cys Ile Gly Gly Pr - #o Leu His Asn Gly Leu          #               255                                                            - Asp Gln Leu Gln Asn Cys Phe Thr Leu Leu Al - #a Glu Ser Leu Phe Gln          #           270                                                                - Leu Arg Gln Gln Leu Glu Lys Leu Gln Glu Gl - #n Ser Thr Lys Met Thr          #       285                                                                    - Tyr Glu Gly Asp Pro Ile Pro Ala Gln Arg Al - #a His Leu Leu Glu Arg          #   300                                                                        - Ala Thr Phe Leu Ile Tyr Asn Leu Phe Lys As - #n Ser Phe Val Val Glu          305                 3 - #10                 3 - #15                 3 -        #20                                                                            - Arg His Ala Cys Met Pro Thr His Pro Gln Ar - #g Pro Met Val Leu Lys          #               335                                                            - Thr Leu Ile Gln Phe Thr Val Lys Leu Arg Le - #u Leu Ile Lys Leu Pro          #           350                                                                - Glu Leu Asn Tyr Gln Val Lys Val Lys Ala Se - #r Ile Asp Lys Asn Val          #       365                                                                    - Ser Thr Leu Ser Asn Arg Arg Phe Val Leu Cy - #s Gly Thr His Val Lys          #   380                                                                        - Ala Met Ser Ser Glu Glu Ser Ser Asn Gly Se - #r Leu Ser Val Glu Leu          385                 3 - #90                 3 - #95                 4 -        #00                                                                            - Asp Ile Ala Thr Gln Gly Asp Glu Val Gln Ty - #r Trp Ser Lys Gly Asn          #               415                                                            - Glu Gly Cys His Met Val Thr Glu Glu Leu Hi - #s Ser Ile Thr Phe Glu          #           430                                                                - Thr Gln Ile Cys Leu Tyr Gly Leu Thr Ile As - #n Leu Glu Thr Ser Ser          #       445                                                                    - Leu Pro Val Val Met Ile Ser Asn Val Ser Gl - #n Leu Pro Asn Ala Trp          #   460                                                                        - Ala Ser Ile Ile Trp Tyr Asn Val Ser Thr As - #n Asp Ser Gln Asn Leu          465                 4 - #70                 4 - #75                 4 -        #80                                                                            - Val Phe Phe Asn Asn Pro Pro Ser Val Thr Le - #u Gly Gln Leu Leu Glu          #               495                                                            - Val Met Ser Trp Gln Phe Ser Ser Tyr Val Gl - #y Arg Gly Leu Asn Ser          #           510                                                                - Glu Gln Leu Asn Met Leu Ala Glu Lys Leu Th - #r Val Gln Ser Asn Tyr          #       525                                                                    - Asn Asp Gly His Leu Thr Trp Ala Lys Phe Cy - #s Lys Glu His Leu Pro          #   540                                                                        - Gly Lys Thr Phe Thr Phe Trp Thr Trp Leu Gl - #u Ala Ile Leu Asp Leu          545                 5 - #50                 5 - #55                 5 -        #60                                                                            - Ile Lys Lys His Ile Leu Pro Leu Trp Ile As - #p Gly Tyr Ile Met Gly          #               575                                                            - Phe Val Ser Lys Glu Lys Glu Arg Leu Leu Le - #u Lys Asp Lys Met Pro          #           590                                                                - Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser Hi - #s Leu Gly Gly Ile Thr          #       605                                                                    - Phe Thr Trp Val Asp Gln Ser Glu Asn Gly Gl - #u Val Arg Phe His Ser          #   620                                                                        - Val Glu Pro Tyr Asn Lys Gly Arg Leu Ser Al - #a Leu Ala Phe Ala Asp          625                 6 - #30                 6 - #35                 6 -        #40                                                                            - Ile Leu Arg Asp Tyr Lys Val Ile Met Ala Gl - #u Asn Ile Pro Glu Asn          #               655                                                            - Pro Leu Lys Tyr Leu Tyr Pro Asp Ile Pro Ly - #s Asp Lys Ala Phe Gly          #           670                                                                - Lys His Tyr Ser Ser Gln Pro Cys Glu Val Se - #r Arg Pro Thr Glu Arg          #       685                                                                    - Gly Asp Lys Gly Tyr Val Pro Ser Val Phe Il - #e Pro Ile Ser Thr Ile          #   700                                                                        - Arg Ser Asp Ser Thr Glu Pro Gln Ser Pro Se - #r Asp Leu Leu Pro Met          705                 7 - #10                 7 - #15                 7 -        #20                                                                            - Ser Pro Ser Ala Tyr Ala Val Leu Arg Glu As - #n Leu Ser Pro Thr Thr          #               735                                                            - Ile Glu Thr Ala Met Asn Ser Pro Tyr Ser Al - #a Glu                          #           745                                                                - (2) INFORMATION FOR SEQ ID NO:11:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 2869 base                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: both                                                         (D) TOPOLOGY: unknown                                                -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Mouse                                                  -    (vii) IMMEDIATE SOURCE:                                                             (A) LIBRARY: splenic/thymi - #c                                                (B) CLONE: Murine 19sf6                                              -     (ix) FEATURE:                                                                      (A) NAME/KEY: CDS                                                              (B) LOCATION: 69..2378                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                 - GCCGCGACCA GCCAGGCCGG CCAGTCGGGC TCAGCCCGGA GACAGTCGAG AC - #CCCTGACT          60                                                                           #CTG GAC ACA CGC TAC     110AC CAG CTG CAG CAG                                          Met Ala Gln Trp Asn G - #ln Leu Gln Gln Leu Asp Thr Arg Tyr           #        10                                                                    - CTG AAG CAG CTG CAC CAG CTG TAC AGC GAC AC - #G TTC CCC ATG GAG CTG           158                                                                           Leu Lys Gln Leu His Gln Leu Tyr Ser Asp Th - #r Phe Pro Met Glu Leu            # 30                                                                           - CGG CAG TTC CTG GCA CCT TGG ATT GAG AGT CA - #A GAC TGG GCA TAT GCA           206                                                                           Arg Gln Phe Leu Ala Pro Trp Ile Glu Ser Gl - #n Asp Trp Ala Tyr Ala            #                 45                                                           - GCC AGC AAA GAG TCA CAT GCC ACG TTG GTG TT - #T CAT AAT CTC TTG GGT           254                                                                           Ala Ser Lys Glu Ser His Ala Thr Leu Val Ph - #e His Asn Leu Leu Gly            #             60                                                               - GAA ATT GAC CAG CAA TAT AGC CGA TTC CTG CA - #A GAG TCC AAT GTC CTC           302                                                                           Glu Ile Asp Gln Gln Tyr Ser Arg Phe Leu Gl - #n Glu Ser Asn Val Leu            #         75                                                                   - TAT CAG CAC AAC CTT CGA AGA ATC AAG CAG TT - #T CTG CAG AGC AGG TAT           350                                                                           Tyr Gln His Asn Leu Arg Arg Ile Lys Gln Ph - #e Leu Gln Ser Arg Tyr            #     90                                                                       - CTT GAG AAG CCA ATG GAA ATT GCC CGG ATC GT - #G GCC CGA TGC CTG TGG           398                                                                           Leu Glu Lys Pro Met Glu Ile Ala Arg Ile Va - #l Ala Arg Cys Leu Trp            #110                                                                           - GAA GAG TCT CGC CTC CTC CAG ACG GCA GCC AC - #G GCA GCC CAG CAA GGG           446                                                                           Glu Glu Ser Arg Leu Leu Gln Thr Ala Ala Th - #r Ala Ala Gln Gln Gly            #               125                                                            - GGC CAG GCC AAC CAC CCA ACA GCC GCC GTA GT - #G ACA GAG AAG CAG CAG           494                                                                           Gly Gln Ala Asn His Pro Thr Ala Ala Val Va - #l Thr Glu Lys Gln Gln            #           140                                                                - ATG TTG GAG CAG CAT CTT CAG GAT GTC CGG AA - #G CGA GTG CAG GAT CTA           542                                                                           Met Leu Glu Gln His Leu Gln Asp Val Arg Ly - #s Arg Val Gln Asp Leu            #       155                                                                    - GAA CAG AAA ATG AAG GTG GTG GAG AAC CTC CA - #G GAC GAC TTT GAT TTC           590                                                                           Glu Gln Lys Met Lys Val Val Glu Asn Leu Gl - #n Asp Asp Phe Asp Phe            #   170                                                                        - AAC TAC AAA ACC CTC AAG AGC CAA GGA GAC AT - #G CAG GAT CTG AAT GGA           638                                                                           Asn Tyr Lys Thr Leu Lys Ser Gln Gly Asp Me - #t Gln Asp Leu Asn Gly            175                 1 - #80                 1 - #85                 1 -        #90                                                                            - AAC AAC CAG TCT GTG ACC AGA CAG AAG ATG CA - #G CAG CTG GAA CAG ATG           686                                                                           Asn Asn Gln Ser Val Thr Arg Gln Lys Met Gl - #n Gln Leu Glu Gln Met            #               205                                                            - CTC ACA GCC CTG GAC CAG ATG CGG AGA AGC AT - #T GTG AGT GAG CTG GCG           734                                                                           Leu Thr Ala Leu Asp Gln Met Arg Arg Ser Il - #e Val Ser Glu Leu Ala            #           220                                                                - GGG CTC TTG TCA GCA ATG GAG TAC GTG CAG AA - #G ACA CTG ACT GAT GAA           782                                                                           Gly Leu Leu Ser Ala Met Glu Tyr Val Gln Ly - #s Thr Leu Thr Asp Glu            #       235                                                                    - GAG CTG GCT GAC TGG AAG AGG CGG CCA GAG AT - #C GCG TGC ATC GGA GGC           830                                                                           Glu Leu Ala Asp Trp Lys Arg Arg Pro Glu Il - #e Ala Cys Ile Gly Gly            #   250                                                                        - CCT CCC AAC ATC TGC CTG GAC CGT CTG GAA AA - #C TGG ATA ACT TCA TTA           878                                                                           Pro Pro Asn Ile Cys Leu Asp Arg Leu Glu As - #n Trp Ile Thr Ser Leu            255                 2 - #60                 2 - #65                 2 -        #70                                                                            - GCA GAA TCT CAA CTT CAG ACC CGC CAA CAA AT - #T AAG AAA CTG GAG GAG           926                                                                           Ala Glu Ser Gln Leu Gln Thr Arg Gln Gln Il - #e Lys Lys Leu Glu Glu            #               285                                                            - CTG CAG CAG AAA GTG TCC TAC AAG GGC GAC CC - #T ATC GTG CAG CAC CGG           974                                                                           Leu Gln Gln Lys Val Ser Tyr Lys Gly Asp Pr - #o Ile Val Gln His Arg            #           300                                                                - CCC ATG CTG GAG GAG AGG ATC GTG GAG CTG TT - #C AGA AAC TTA ATG AAG          1022                                                                           Pro Met Leu Glu Glu Arg Ile Val Glu Leu Ph - #e Arg Asn Leu Met Lys            #       315                                                                    - AGT GCC TTC GTG GTG GAG CGG CAG CCC TGC AT - #G CCC ATG CAC CCG GAC          1070                                                                           Ser Ala Phe Val Val Glu Arg Gln Pro Cys Me - #t Pro Met His Pro Asp            #   330                                                                        - CGG CCC TTA GTC ATC AAG ACT GGT GTC CAG TT - #T ACC ACG AAA GTC AGG          1118                                                                           Arg Pro Leu Val Ile Lys Thr Gly Val Gln Ph - #e Thr Thr Lys Val Arg            335                 3 - #40                 3 - #45                 3 -        #50                                                                            - TTG CTG GTC AAA TTT CCT GAG TTG AAT TAT CA - #G CTT AAA ATT AAA GTG          1166                                                                           Leu Leu Val Lys Phe Pro Glu Leu Asn Tyr Gl - #n Leu Lys Ile Lys Val            #               365                                                            - TGC ATT GAT AAA GAC TCT GGG GAT GTT GCT GC - #C CTC AGA GGG TCT CGG          1214                                                                           Cys Ile Asp Lys Asp Ser Gly Asp Val Ala Al - #a Leu Arg Gly Ser Arg            #           380                                                                - AAA TTT AAC ATT CTG GGC ACG AAC ACA AAA GT - #G ATG AAC ATG GAG GAG          1262                                                                           Lys Phe Asn Ile Leu Gly Thr Asn Thr Lys Va - #l Met Asn Met Glu Glu            #       395                                                                    - TCT AAC AAC GGC AGC CTG TCT GCA GAG TTC AA - #G CAC CTG ACC CTT AGG          1310                                                                           Ser Asn Asn Gly Ser Leu Ser Ala Glu Phe Ly - #s His Leu Thr Leu Arg            #   410                                                                        - GAG CAG AGA TGT GGG AAT GGA GGC CGT GCC AA - #T TGT GAT GCC TCC TTG          1358                                                                           Glu Gln Arg Cys Gly Asn Gly Gly Arg Ala As - #n Cys Asp Ala Ser Leu            415                 4 - #20                 4 - #25                 4 -        #30                                                                            - ATC GTG ACT GAG GAG CTG CAC CTG ATC ACC TT - #C GAG ACT GAG GTG TAC          1406                                                                           Ile Val Thr Glu Glu Leu His Leu Ile Thr Ph - #e Glu Thr Glu Val Tyr            #               445                                                            - CAC CAA GGC CTC AAG ATT GAC CTA GAG ACC CA - #C TCC TTG CCA GTT GTG          1454                                                                           His Gln Gly Leu Lys Ile Asp Leu Glu Thr Hi - #s Ser Leu Pro Val Val            #           460                                                                - GTG ATC TCC AAC ATC TGT CAG ATG CCA AAT GC - #T TGG GCA TCA ATC CTG          1502                                                                           Val Ile Ser Asn Ile Cys Gln Met Pro Asn Al - #a Trp Ala Ser Ile Leu            #       475                                                                    - TGG TAT AAC ATG CTG ACC AAT AAC CCC AAG AA - #C GTG AAC TTC TTC ACT          1550                                                                           Trp Tyr Asn Met Leu Thr Asn Asn Pro Lys As - #n Val Asn Phe Phe Thr            #   490                                                                        - AAG CCG CCA ATT GGA ACC TGG GAC CAA GTG GC - #C GAG GTG CTC AGC TGG          1598                                                                           Lys Pro Pro Ile Gly Thr Trp Asp Gln Val Al - #a Glu Val Leu Ser Trp            495                 5 - #00                 5 - #05                 5 -        #10                                                                            - CAG TTC TCG TCC ACC ACC AAG CGA GGG CTG AG - #C ATC GAG CAG CTG ACA          1646                                                                           Gln Phe Ser Ser Thr Thr Lys Arg Gly Leu Se - #r Ile Glu Gln Leu Thr            #               525                                                            - ACG CTG GCT GAG AAG CTC CTA GGG CCT GGT GT - #G AAC TAC TCA GGG TGT          1694                                                                           Thr Leu Ala Glu Lys Leu Leu Gly Pro Gly Va - #l Asn Tyr Ser Gly Cys            #           540                                                                - CAG ATC ACA TGG GCT AAA TTC TGC AAA GAA AA - #C ATG GCT GGC AAG GGC          1742                                                                           Gln Ile Thr Trp Ala Lys Phe Cys Lys Glu As - #n Met Ala Gly Lys Gly            #       555                                                                    - TTC TCC TTC TGG GTC TGG CTA GAC AAT ATC AT - #C GAC CTT GTG AAA AAG          1790                                                                           Phe Ser Phe Trp Val Trp Leu Asp Asn Ile Il - #e Asp Leu Val Lys Lys            #   570                                                                        - TAT ATC TTG GCC CTT TGG AAT GAA GGG TAC AT - #C ATG GGT TTC ATC AGC          1838                                                                           Tyr Ile Leu Ala Leu Trp Asn Glu Gly Tyr Il - #e Met Gly Phe Ile Ser            575                 5 - #80                 5 - #85                 5 -        #90                                                                            - AAG GAG CGG GAG CGG GCC ATC CTA AGC ACA AA - #G CCC CCG GGC ACC TTC          1886                                                                           Lys Glu Arg Glu Arg Ala Ile Leu Ser Thr Ly - #s Pro Pro Gly Thr Phe            #               605                                                            - CTA CTG CGC TTC AGC GAG AGC AGC AAA GAA GG - #A GGG GTC ACT TTC ACT          1934                                                                           Leu Leu Arg Phe Ser Glu Ser Ser Lys Glu Gl - #y Gly Val Thr Phe Thr            #           620                                                                - TGG GTG GAA AAG GAC ATC AGT GGC AAG ACC CA - #G ATC CAG TCT GTA GAG          1982                                                                           Trp Val Glu Lys Asp Ile Ser Gly Lys Thr Gl - #n Ile Gln Ser Val Glu            #       635                                                                    - CCA TAC ACC AAG CAG CAG CTG AAC AAC ATG TC - #A TTT GCT GAA ATC ATC          2030                                                                           Pro Tyr Thr Lys Gln Gln Leu Asn Asn Met Se - #r Phe Ala Glu Ile Ile            #   650                                                                        - ATG GGC TAT AAG ATC ATG GAT GCG ACC AAC AT - #C CTG GTG TCT CCA CTT          2078                                                                           Met Gly Tyr Lys Ile Met Asp Ala Thr Asn Il - #e Leu Val Ser Pro Leu            655                 6 - #60                 6 - #65                 6 -        #70                                                                            - GTC TAC CTC TAC CCC GAC ATT CCC AAG GAG GA - #G GCA TTT GGA AAG TAC          2126                                                                           Val Tyr Leu Tyr Pro Asp Ile Pro Lys Glu Gl - #u Ala Phe Gly Lys Tyr            #               685                                                            - TGT AGG CCC GAG AGC CAG GAG CAC CCC GAA GC - #C GAC CCA GGT AGT GCT          2174                                                                           Cys Arg Pro Glu Ser Gln Glu His Pro Glu Al - #a Asp Pro Gly Ser Ala            #           700                                                                - GCC CCG TAC CTG AAG ACC AAG TTC ATC TGT GT - #G ACA CCA ACG ACC TGC          2222                                                                           Ala Pro Tyr Leu Lys Thr Lys Phe Ile Cys Va - #l Thr Pro Thr Thr Cys            #       715                                                                    - AGC AAT ACC ATT GAC CTG CCG ATG TCC CCC CG - #C ACT TTA GAT TCA TTG          2270                                                                           Ser Asn Thr Ile Asp Leu Pro Met Ser Pro Ar - #g Thr Leu Asp Ser Leu            #   730                                                                        - ATG CAG TTT GGA AAT AAC GGT GAA GGT GCT GA - #G CCC TCA GCA GGA GGG          2318                                                                           Met Gln Phe Gly Asn Asn Gly Glu Gly Ala Gl - #u Pro Ser Ala Gly Gly            735                 7 - #40                 7 - #45                 7 -        #50                                                                            - CAG TTT GAG TCG CTC ACG TTT GAC ATG GAT CT - #G ACC TCG GAG TGT GCT          2366                                                                           Gln Phe Glu Ser Leu Thr Phe Asp Met Asp Le - #u Thr Ser Glu Cys Ala            #               765                                                            - ACC TCC CCC ATG TGAGGAGCTG AAACCAGAAG CTGCAGAGAC GT - #GACTTGAG              2418                                                                           Thr Ser Pro Met                                                                            770                                                                - ACACCTGCCC CGTGCTCCAC CCCTAAGCAG CCGAACCCCA TATCGTCTGA AA - #CTCCTAAC        2478                                                                           - TTTGTGGTTC CAGATTTTTT TTTTTAATTT CCTACTTCTG CTATCTTTGG GC - #AATCTGGG        2538                                                                           - CACTTTTTAA AAGAGAGAAA TGAGTGAGTG TGGGTGATAA ACTGTTATGT AA - #AGAGGAGA        2598                                                                           - GACCTCTGAG TCTGGGGATG GGGCTGAGAG CAGAAGGGAG GCAAAGGGGA AC - #ACCTCCTG        2658                                                                           - TCCTGCCCGC CTGCCCTCCT TTTTCAGCAG CTCGGGGGTT GGTTGTTAGA CA - #AGTGCCTC        2718                                                                           - CTGGTGCCCA TGGCTACCTG TTGCCCCACT CTGTGAGCTG ATACCCCATT CT - #GGGAACTC        2778                                                                           - CTGGCTCTGC ACTTTCAACC TTGCTAATAT CCACATAGAA GCTAGGACTA AG - #CCCAGGAG        2838                                                                           #        2869      AAAA AAAAAAAAAA A                                           - (2) INFORMATION FOR SEQ ID NO:12:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 770 amino                                                          (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: protein                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                 - Met Ala Gln Trp Asn Gln Leu Gln Gln Leu As - #p Thr Arg Tyr Leu Lys          #                 15                                                           - Gln Leu His Gln Leu Tyr Ser Asp Thr Phe Pr - #o Met Glu Leu Arg Gln          #             30                                                               - Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Tr - #p Ala Tyr Ala Ala Ser          #         45                                                                   - Lys Glu Ser His Ala Thr Leu Val Phe His As - #n Leu Leu Gly Glu Ile          #     60                                                                       - Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Se - #r Asn Val Leu Tyr Gln          # 80                                                                           - His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gl - #n Ser Arg Tyr Leu Glu          #                 95                                                           - Lys Pro Met Glu Ile Ala Arg Ile Val Ala Ar - #g Cys Leu Trp Glu Glu          #           110                                                                - Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Al - #a Gln Gln Gly Gly Gln          #       125                                                                    - Ala Asn His Pro Thr Ala Ala Val Val Thr Gl - #u Lys Gln Gln Met Leu          #   140                                                                        - Glu Gln His Leu Gln Asp Val Arg Lys Arg Va - #l Gln Asp Leu Glu Gln          145                 1 - #50                 1 - #55                 1 -        #60                                                                            - Lys Met Lys Val Val Glu Asn Leu Gln Asp As - #p Phe Asp Phe Asn Tyr          #               175                                                            - Lys Thr Leu Lys Ser Gln Gly Asp Met Gln As - #p Leu Asn Gly Asn Asn          #           190                                                                - Gln Ser Val Thr Arg Gln Lys Met Gln Gln Le - #u Glu Gln Met Leu Thr          #       205                                                                    - Ala Leu Asp Gln Met Arg Arg Ser Ile Val Se - #r Glu Leu Ala Gly Leu          #   220                                                                        - Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Le - #u Thr Asp Glu Glu Leu          225                 2 - #30                 2 - #35                 2 -        #40                                                                            - Ala Asp Trp Lys Arg Arg Pro Glu Ile Ala Cy - #s Ile Gly Gly Pro Pro          #               255                                                            - Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Il - #e Thr Ser Leu Ala Glu          #           270                                                                - Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Ly - #s Leu Glu Glu Leu Gln          #       285                                                                    - Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Va - #l Gln His Arg Pro Met          #   300                                                                        - Leu Glu Glu Arg Ile Val Glu Leu Phe Arg As - #n Leu Met Lys Ser Ala          305                 3 - #10                 3 - #15                 3 -        #20                                                                            - Phe Val Val Glu Arg Gln Pro Cys Met Pro Me - #t His Pro Asp Arg Pro          #               335                                                            - Leu Val Ile Lys Thr Gly Val Gln Phe Thr Th - #r Lys Val Arg Leu Leu          #           350                                                                - Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Ly - #s Ile Lys Val Cys Ile          #       365                                                                    - Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Ar - #g Gly Ser Arg Lys Phe          #   380                                                                        - Asn Ile Leu Gly Thr Asn Thr Lys Val Met As - #n Met Glu Glu Ser Asn          385                 3 - #90                 3 - #95                 4 -        #00                                                                            - Asn Gly Ser Leu Ser Ala Glu Phe Lys His Le - #u Thr Leu Arg Glu Gln          #               415                                                            - Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys As - #p Ala Ser Leu Ile Val          #           430                                                                - Thr Glu Glu Leu His Leu Ile Thr Phe Glu Th - #r Glu Val Tyr His Gln          #       445                                                                    - Gly Leu Lys Ile Asp Leu Glu Thr His Ser Le - #u Pro Val Val Val Ile          #   460                                                                        - Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Al - #a Ser Ile Leu Trp Tyr          465                 4 - #70                 4 - #75                 4 -        #80                                                                            - Asn Met Leu Thr Asn Asn Pro Lys Asn Val As - #n Phe Phe Thr Lys Pro          #               495                                                            - Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Va - #l Leu Ser Trp Gln Phe          #           510                                                                - Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Gl - #u Gln Leu Thr Thr Leu          #       525                                                                    - Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Ty - #r Ser Gly Cys Gln Ile          #   540                                                                        - Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Al - #a Gly Lys Gly Phe Ser          545                 5 - #50                 5 - #55                 5 -        #60                                                                            - Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Le - #u Val Lys Lys Tyr Ile          #               575                                                            - Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gl - #y Phe Ile Ser Lys Glu          #           590                                                                - Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pr - #o Gly Thr Phe Leu Leu          #       605                                                                    - Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Va - #l Thr Phe Thr Trp Val          #   620                                                                        - Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gl - #n Ser Val Glu Pro Tyr          625                 6 - #30                 6 - #35                 6 -        #40                                                                            - Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Al - #a Glu Ile Ile Met Gly          #               655                                                            - Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Va - #l Ser Pro Leu Val Tyr          #           670                                                                - Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Ph - #e Gly Lys Tyr Cys Arg          #       685                                                                    - Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pr - #o Gly Ser Ala Ala Pro          #   700                                                                        - Tyr Leu Lys Thr Lys Phe Ile Cys Val Thr Pr - #o Thr Thr Cys Ser Asn          705                 7 - #10                 7 - #15                 7 -        #20                                                                            - Thr Ile Asp Leu Pro Met Ser Pro Arg Thr Le - #u Asp Ser Leu Met Gln          #               735                                                            - Phe Gly Asn Asn Gly Glu Gly Ala Glu Pro Se - #r Ala Gly Gly Gln Phe          #           750                                                                - Glu Ser Leu Thr Phe Asp Met Asp Leu Thr Se - #r Glu Cys Ala Thr Ser          #       765                                                                    - Pro Met                                                                          770                                                                        - (2) INFORMATION FOR SEQ ID NO:13:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 24 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Homo sapi - #ens                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                 #                24ARAT YATT                                                   - (2) INFORMATION FOR SEQ ID NO:14:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 21 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Homo sapi - #ens                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                 #21                AYAT G                                                      - (2) INFORMATION FOR SEQ ID NO:15:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 18 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Homo sapi - #ens                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                 #  18              AR                                                          - (2) INFORMATION FOR SEQ ID NO:16:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 20 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (vi) ORIGINAL SOURCE:                                                              (A) ORGANISM: Homo sapi - #ens                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                 # 20               GYAA                                                        - (2) INFORMATION FOR SEQ ID NO:17:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 25 base                                                            (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: cDNA                                                 -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                 #               25 CCAG AAAAG                                                  - (2) INFORMATION FOR SEQ ID NO:18:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 15 amino                                                           (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -      (v) FRAGMENT TYPE: internal                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                 - Leu Asp Gly Pro Lys Gly Thr Gly Tyr Ile Ly - #s Thr Glu Leu Ile              #                15                                                            - (2) INFORMATION FOR SEQ ID NO:19:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 6 amino                                                            (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -      (v) FRAGMENT TYPE: internal                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                 - Gly Tyr Ile Lys Thr Glu                                                      1               5                                                              - (2) INFORMATION FOR SEQ ID NO:20:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 14 amino                                                           (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -      (v) FRAGMENT TYPE: internal                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                 - Lys Val Asn Leu Gln Glu Arg Arg Lys Tyr Le - #u Lys His Arg                  #                10                                                            - (2) INFORMATION FOR SEQ ID NO:21:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 11 amino                                                           (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -      (v) FRAGMENT TYPE: internal                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                 - Glu Pro Gln Tyr Glu Glu Ile Pro Ile Tyr Le - #u                              #                10                                                            - (2) INFORMATION FOR SEQ ID NO:22:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 105 amino                                                          (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -      (v) FRAGMENT TYPE: internal                                             -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: Src                                                       -      (x) PUBLICATION INFORMATION:                                                      (A) AUTHORS: Waksman, e - #t al.                                               (C) JOURNAL: Nature                                                            (D) VOLUME: 358                                                                (F) PAGES: 646-653                                                             (G) DATE: 1992                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                 - Ala Glu Glu Trp Tyr Phe Gly Lys Ile Thr Ar - #g Arg Glu Ser Glu Arg          #                15                                                            - Leu Leu Leu Asn Pro Glu Asn Pro Arg Gly Th - #r Phe Leu Val Arg Glu          #            30                                                                - Ser Glu Thr Thr Lys Gly Ala Tyr Cys Leu Se - #r Val Ser Asp Phe Phe          #        45                                                                    - Asp Asn Ala Lys Gly Leu Asn Val Lys His Ty - #r Lys Ile Arg Lys Leu          #    60                                                                        - Asp Ser Gly Gly Phe Tyr Ile Thr Ser Arg Th - #r Gln Phe Ser Ser Leu          #80                                                                            - Gln Gln Leu Val Ala Tyr Tyr Ser Lys His Al - #a Asp Gly Leu Cys His          #                95                                                            - Arg Leu Thr Asn Val Cys Pro Thr Ser                                          #           105                                                                - (2) INFORMATION FOR SEQ ID NO:23:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 99 amino                                                           (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -      (v) FRAGMENT TYPE: internal                                             -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: Abl                                                       -      (x) PUBLICATION INFORMATION:                                            #et al.   (A) AUTHORS: Overduin,                                                         (C) JOURNAL: Proc. Natl - #. Acad. Sci. U.S.A.                                 (D) VOLUME: 89                                                                 (F) PAGES: 11673-11677                                                         (G) DATE: 1992                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                 - Glu Lys His Ser Trp Tyr His Gly Pro Val Se - #r Arg Asn Ala Ala Glu          #                15                                                            - Tyr Leu Leu Ser Ser Gly Ile Asn Gly Ser Ph - #e Leu Val Arg Glu Ser          #            30                                                                - Asp Arg Arg Pro Gly Gln Arg Ser Ile Ser Le - #u Arg Tyr Glu Glu Gly          #        45                                                                    - Arg Val Tyr His Tyr Arg Ile Asn Thr Ala Se - #r Asp Gly Lys Leu Tyr          #    60                                                                        - Val Ser Ser Glu Ser Arg Phe Asn Thr Leu Al - #a Glu Leu Val His His          #80                                                                            - His Ser Thr Val Ala Asp Gly Leu Ile Thr Th - #r Leu His Tyr Pro Ala          #                95                                                            - Pro Lys Arg                                                                  - (2) INFORMATION FOR SEQ ID NO:24:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 102 amino                                                          (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -      (v) FRAGMENT TYPE: internal                                             -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: Lck                                                       -      (x) PUBLICATION INFORMATION:                                            #al.      (A) AUTHORS: Eck, et                                                           (C) JOURNAL: Nature                                                            (D) VOLUME: 362                                                                (F) PAGES: 87-91                                                               (G) DATE: 1993                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                 - Trp Phe Phe Lys Asn Leu Ser Arg Lys Asp Al - #a Glu Arg Gln Leu Leu          #                15                                                            - Ala Pro Gly Asn Thr His Gly Ser Phe Leu Il - #e Arg Glu Ser Glu Ser          #            30                                                                - Thr Ala Gly Ser Phe Ser Leu Ser Val Arg As - #p Asp Phe Asp Gln Asn          #        45                                                                    - Gln Gly Glu Val Val Lys His Tyr Lys Ile Ar - #g Asn Leu Asp Asn Gly          #    60                                                                        - Gly Phe Tyr Ile Ser Pro Arg Ile Thr Phe Pr - #o Gly Leu His Asp Leu          #80                                                                            - Val Arg His Tyr Thr Asn Ala Ser Asp Gly Le - #u Cys Thr Arg Leu Ser          #                95                                                            - Arg Pro Cys Gln Thr Gln                                                                  100                                                                - (2) INFORMATION FOR SEQ ID NO:25:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 99 amino                                                           (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              -    (iii) HYPOTHETICAL: NO                                                    -     (iv) ANTI-SENSE: NO                                                      -      (v) FRAGMENT TYPE: internal                                             -    (vii) IMMEDIATE SOURCE:                                                             (B) CLONE: p85[alpha]N                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                 - Gln Asp Ala Glu Trp Tyr Trp Gly Asp Ile Se - #r Arg Glu Glu Val Asn          #                15                                                            - Glu Lys Leu Arg Asp Thr Ala Asp Gly Thr Ph - #e Leu Val Arg Asp Ala          #            30                                                                - Ser Thr Lys Met His Gly Asp Tyr Thr Leu Th - #r Leu Arg Lys Gly Gly          #        45                                                                    - Asn Asn Lys Leu Ile Lys Ile Phe His Arg As - #p Gly Lys Tyr Gly Phe          #    60                                                                        - Ser Asp Pro Leu Thr Phe Asn Ser Val Val Gl - #u Leu Ile Asn His Tyr          #80                                                                            - Arg His Glu Ser Leu Ala Gln Tyr Asn Pro Ly - #s Leu Asp Val Lys Leu          #                95                                                            - Leu Tyr Pro                                                                  __________________________________________________________________________ 

What is claimed is:
 1. A recombinant DNA molecule comprising a DNA sequence encoding a receptor recognition factor (RRF), Stat3, having the amino acid sequence of SEQ ID NO:12.
 2. The recombinant DNA molecule of claim 1 wherein the DNA sequence is the coding region of SEQ ID NO:11.
 3. The recombinant DNA molecule of claim 1 wherein said DNA sequence is operatively linked to an expression control sequence.
 4. An expression vector containing the recombinant DNA molecule of claim
 3. 5. A method of expressing a recombinant receptor recognition factor in a cell containing the expression vector of claim 4 comprising culturing the cell in an appropriate cell culture medium under conditions that provide for expression of the receptor recognition factor by the cell.
 6. The method of claim 5 further comprising the step of purifying the recombinant receptor recognition factor.
 7. The method of claim 6 wherein the receptor recognition factor has the amino acid sequence of SEQ ID NO:12.
 8. An isolated nucleic acid encoding a receptor recognition factor (RRF), Stat3, having the amino acid of SEQ ID NO:12.
 9. A recombinant DNA molecule comprising 25 contiguous nucleotides from a nucleic acid encoding a Stat3 receptor recognition factor, wherein said nucleic acid has the nucleotide sequence of the coding region of SEQ ID NO:11.
 10. The recombinant DNA molecule of claim 9 that is operatively linked to an expression control sequence.
 11. An expression vector containing the recombinant DNA molecule of claim
 10. 12. A recombinant DNA molecule encoding a Stat3, wherein the recombinant DNA molecule hybridizes under standard hybridization conditions of 5×SSC and 65° C. to a nucleic acid complementary to the nucleotide sequence of SEQ ID NO:11 and wherein said recombinant DNA molecule encodes a polypeptide containing a tyrosyl residue that is phosphorylated when said polypeptide is expressed in a cell treated with IL-6; wherein when said cell expresses a protein having the amino acid sequence of SEQ ID NO: 12 said protein is phosphorylated in response to IL-6 treatment.
 13. The recombinant DNA molecule of claim 12 that is operatively linked to an expression control sequence.
 14. An expression vector containing the recombinant DNA molecule of claim
 13. 15. A method of expressing the recombinant DNA molecule of claim 14 in a cell containing the expression vector comprising culturing the cell in an appropriate cell culture medium under conditions that provide for expression of the recombinant DNA molecule by the cell.
 16. The method of claim 15 further comprising the step of purifying a protein product of the expression of said recombinant DNA molecule. 